U.S. patent application number 10/572189 was filed with the patent office on 2007-04-05 for secretion of proteins from yeasts.
This patent application is currently assigned to BASF Aktiengesellscaft. Invention is credited to Kai Ostermann, Gerhard Rodel.
Application Number | 20070077619 10/572189 |
Document ID | / |
Family ID | 34352870 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070077619 |
Kind Code |
A1 |
Ostermann; Kai ; et
al. |
April 5, 2007 |
Secretion of proteins from yeasts
Abstract
The present invention relates to expression constructs
encompassing the nucleic acid sequence coding for a shuttle peptide
construct processable by yeast cells; to corresponding expression
vectors comprising such constructs; to methods carried out with the
aid thereof for recombinant preparation of target proteins; to
hosts transformed therewith; to shuttle peptides and nucleic acid
sequences coding therefor; to nucleic acid sequences coding for
such shuttle peptides fused to a foreign protein; to hydrophobin
proteins prepared using shuttle peptides of this kind and to the
use of hydrophobins for the coating of objects such as, for
example, leather.
Inventors: |
Ostermann; Kai; (Dresden,
DE) ; Rodel; Gerhard; (Karlsfeld, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
BASF Aktiengesellscaft
Ludwigshafen
DE
D-67056
|
Family ID: |
34352870 |
Appl. No.: |
10/572189 |
Filed: |
September 15, 2004 |
PCT Filed: |
September 15, 2004 |
PCT NO: |
PCT/EP04/10348 |
371 Date: |
March 15, 2006 |
Current U.S.
Class: |
435/69.1 ;
435/254.2; 435/483; 510/320; 530/359; 536/23.7 |
Current CPC
Class: |
C12N 15/815
20130101 |
Class at
Publication: |
435/069.1 ;
435/254.2; 435/483; 530/359; 536/023.7; 510/320 |
International
Class: |
C11D 3/386 20060101
C11D003/386; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 15/74 20060101 C12N015/74; C07K 14/39 20060101
C07K014/39 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 2003 |
DE |
10342794.5 |
Claims
1. An expression construct, which comprises a nucleic acid sequence
coding for a shuttle peptide construct which is processable by
yeast cells, has the formula (Sig-SP), and comprises in 5'-3'
orientation the nucleic acid sequences coding for a) a signal
peptide (Sig) processably linked to b) at least one shuttle peptide
(SP) secretable by said yeast cells.
2. The expression construct as claimed in claim 1, wherein the
shuttle peptide construct (Sig-SP) is derived from polypeptide
processed by yeasts of the genus Schizosaccharomyces.
3. The expression construct as claimed in claim 1, wherein the
shuttle peptide construct (Sig-SP) is derived from a pheromone
pre-protein of a yeast, said pheromone (Pher) being derivable from
the pre-protein and secretable by N- and C-terminal processing.
4. The expression construct as claimed in claim 3, wherein the
signal polypeptide (Sig) is the proteolytically removable native
signal polypeptide of the pheromone pre-protein.
5. The expression construct as claimed in claim 4, wherein the
C-terminally processed pheromone (Pher) comprises a C-terminal
protease cleavage site.
6. The expression construct as claimed in claim 1, further
comprising a nucleic acid sequence coding for a homologous or
heterologous target protein (Targ) processably linked to the
C-terminus of the shuttle peptide construct (Sig-SP).
7. The expression construct as claimed in claim 1 comprising a
nucleic acid sequence coding for a fusion protein which is
processable by yeast cells and has the formula
Sig-L1.sub.n-Pher-L2.sub.m-Targ in which Sig is signal polypeptide,
Pher is processed pheromone, and Targ is target protein, L1 and L2
are processable linkers and n and m are independently of one
another, and is 0 or 1.
8. The expression construct as claimed in claim 1, wherein the
nucleic acid sequence coding for the shuttle peptide construct
(Sig-SP) comprises a signal polypeptide-coding sequence according
to SEQ ID NO: 3 or a functional equivalent thereof which is
operatively linked to the nucleic acid sequence according to SEQ ID
NO: 5, coding for a mature pheromone protein (P factor), or to a
functional equivalent thereof.
9. The expression construct as claimed in claim 1, wherein the
nucleic acid sequence coding for the shuttle peptide construct
comprises a sequence according to SEQ ID NO: 1, optionally extended
at the 3' end by the sequence coding for a target protein
(Targ).
10. The expression construct as claimed in claim 6, wherein the
target protein is a hydrophobin, in particular a class I
hydrophobin.
11. The expression construct as claimed in claim 10, wherein the
hydrophobin is selected from the group consisting of SEQ ID NO: 14
(DewA), SEQ ID NO: 19 (RdlA), SEQ ID NO: 20 (RdlB), SEQ ID NO: 21
(HYP1) and SEQ ID NO: 22 (HYP4), or is encoded by a nucleic acid
sequence according to SEQ ID NO: 13.
12. An expression vector comprising the expression construct as
claimed in claim 1 which is operatively linked to at least one
regulatory nucleic acid sequence.
13. A recombinant microorganism, comprising at least one expression
construct as claimed in claim 1 stably integrated into the host
genome.
14. The microorganism as claimed in claim 13, selected from among
yeasts.
15. The microorganism as claimed in claim 14, selected from among
yeasts of the genus Schizosaccharomyces.
16. A shuttle peptide construct, processable by yeast cells and
derived from a pheromone pre-protein of a yeast, wherein the
pheromone is derivable from said pre-protein and secretable by N-
and C-terminal processing.
17. The shuttle peptide construct as claimed in claim 16,
comprising a signal polypeptide N-terminally processably linked to
the C-terminally processed pheromone polypeptide.
18. The shuttle peptide construct as claimed in claim 17, wherein
the signal polypeptide is the proteolytically removable native
signal polypeptide of the pheromone pre-protein.
19. The shuttle peptide construct as claimed in claim 17, wherein
the C-terminally processed pheromone polypeptide comprises the
C-terminal protease cleavage site.
20. The shuttle peptide construct as claimed in claim 16 comprising
an amino acid sequence as defined in SEQ ID NO: 2 or a functional
equivalent thereof.
21. A method for recombinant preparation of a target protein, which
comprises culturing the microorganism as claimed in claim 13,
expressing the nucleic acid sequence encoding said target protein
and isolating the target protein secreted into the culture
medium.
22. The method as claimed in claim 21, wherein the target protein
is a hydrophobin.
23. A nucleic acid, coding for the shuttle peptide construct as
claimed in claim 16.
24. A nucleic acid coding for the expression construct as claimed
in claim 1.
25. A hydrophobin obtained by the method as claimed in claim
22.
26-28. (canceled)
29. A method of treating the surface of an object comprising
obtaining a hydrophobin by the method according to claim 22, and
treating the surface of the object with the hydrophobin.
30. The method according to claim 29, wherein the object is
selected from the group consisting of glass, fibers, fabrics,
leather, painted objects, films and facades.
31. A method of treating the surface of fibers, fabrics and
leather, wherein the method comprises obtaining a class I
hydrophobin or a hydrophobin as defined in claim 11, and treating
the surface of the fibers, fabrics and leather with the
hydrophobin.
32. The expression construct of claim 2, wherein the yeast is S.
pombe.
33. The microorganism of claim 15, wherein the yeast is S. pombe.
Description
[0001] The present invention relates to expression constructs
encompassing the nucleic acid sequence coding for a shuttle peptide
construct processable by yeast cells; to corresponding expression
vectors comprising such constructs; to methods carried out with the
aid thereof for recombinant preparation of target proteins; to
hosts transformed therewith; to shuttle peptides and nucleic acid
sequences coding therefor; to nucleic acid sequences coding for
such shuttle peptides fused to a foreign protein; to hydrophobin
proteins prepared using shuttle peptides of this kind and to the
use of hydrophobins for the coating of objects such as, for
example, leather.
PRIOR ART
a) Expression in Yeasts
[0002] Yeasts are widely used as hosts for heterologous protein
expression. The reason for this is that a yeast expression system
has several advantages, since yeasts can grow at a higher density
compared to bacteria and other eukaryotic cells and are capable of
protein glycosylation and posttranslational modification. Moreover,
the products produced and secreted by yeasts can be purified in a
simple manner, because yeasts are highly resistant to cell lysis
and the growth medium usually contains low amounts of foreign
protein. In addition, yeasts can grow faster than other eukaryotic
cells at high density on inexpensive nutrient media.
[0003] Numerous different approaches for expressing and secreting
heterologous proteins in yeasts can be found in the prior art.
Thus, for example, U.S. Pat. No. 5,642,487 describes a method for
recombinant production of proteins in yeasts, which comprises
transforming yeast with an expression cassette coding for a
structural element which encodes a leader sequence of an animal
peptide neurohormone, an adaptor sequence producing an
.alpha.-helical structure, a processing signal and a structural
gene.
[0004] It is furthermore known from the prior art to use regulatory
elements of the gene of the .alpha.-factor, a pheromone produced by
yeasts, for controlling expression of heterologous proteins in
yeasts. Thus, for example, .alpha.-factor signal leader peptide
sequences were used for expressing heterologous proteins (cf. e.g.
U.S. Pat. No. 5,010,182).
[0005] Moreover, the published US patent application US
2003/0077831 discloses an expression vector for expressing
heterologous proteins in yeasts which encompasses, flanked by
suitable transcription start and translation start and termination
sequences, the coding sequence for a hybrid precursor polypeptide
whose elements comprise the signal peptide and the leader peptide
of a protein secreted by yeasts and also a heterologous protein
flanked by N-terminal and C-terminal propeptide sequences of said
heterologous protein.
b) Hydrophobins
[0006] Hydrophobins are small cystein-rich proteins comprising
approximately 100 amino acid residues and having interesting
technical propterties. They are capable of rendering hydrophobic
surfaces hydrophilic. They make hydrophilic surfaces become
hydrophobic.
[0007] However, there are a number of patents on hydrophobins and
application thereof: thus, for example, WO-A-96/41882 describes
hydrophobins from edible fungi (cf. SEQ ID NO:21 and 22).
WO-A-00/58342 relates to purification of hydrophobin-containing
fusion proteins by phase extraction. WO-A-01/57066 describes
stabilization, solubilization and, related thereto, improved
application of hydrophobins due to sulfite treatment. WO-A-01/57076
describes purification of hydrophobin via adsorption to Teflon
beads and elution by means of a detergent such as Tween at low
temperatures. WO-A-01/57528 describes fixing hydrophobins to
surfaces by applying Tween and temperatures up to 85 degrees
Celsius.
[0008] WO-A-01/74864 describes untypical hydrophobins (only one
disulfide bridge), referred to as RdlA and RdlB (cf. SEQ ID NO:19
and 20), of filamentous bacteria, in particular Streptomyces sp.
The hydrophobin is used for surface treatment of various objects
such as windows, contact lenses, vehicle bodies. It is furthermore
suggested to produce the proteins described there in a recombinant
host which releases said proteins into the medium. After removing
the host, the hydrophobin-containing medium is supposedly suitable
for surface coating. Experimental evidence of actual expression and
secretion is not provided.
SUMMARY OF THE INVENTION
[0009] It is an object of the present invention to provide means
which make it possible to secrete homologous or, in particular,
heterologous proteins expressed in yeast, in particular
Schizosaccharomyces pombe, from the yeast cells into the
surrounding medium. In particular, means should be provided which
enable recombinantly produced hydrophobin to be secreted from the
host cell.
[0010] We have found that this object is achieved by providing an
expression construct which comprises the nucleic acid sequence
coding for a shuttle peptide construct which is processable by
yeast cells, has the formula (Sig-SP), and comprises in 5'-3'
orientation the nucleic acid sequences coding for [0011] a) a
signal peptide (Sig) processably linked to [0012] b) at least one
shuttle peptide (SP) secretable by said yeast cells.
[0013] Achieving the above object is illustrated in a model way by
the example of the Aspergillus nidulans hydrophobin DewA (mature
protein according to SEQ ID NO:14 with coding sequence according to
SEQ ID NO:13; pre-protein with signal sequence: SEQ ID NO:16 with
coding nucleic acid sequence according to SEQ ID NO:15) as
heterologous target protein (Targ). This protein is a
representative of class I hydrophobins, i.e. of secreted fungal
coat proteins capable of self-assembling.
[0014] In particular, the DNA sequence (SEQ ID NO: 13) coding for
the target protein (DewA) is fused to the 3'-terminal end of the
DNA sequence (SEQ ID NO:5 for mature P factor) coding for an S.
pombe peptide pheromone (P factor; amino acid sequence according to
SEQ ID NO:6 for mature P factor). The resulting fusion protein
comprises all signal sequences required for secreting said
pheromone and the target protein fused thereto, in particular the
removable signal peptide (SEQ ID NO:4). Secretion involves
proteolytic processing of the fusion protein. As a consequence, the
pheromone (P factor) (SEQ ID NO:6) and the target protein
(hydrophobin; SEQ ID NO: 14) are secreted separately into the
medium.
[0015] The finding of the invention is surprising insofar as the
actual regulatory elements of the P-factor pre-protein (N-terminal
of the mature pheromone) are evidently not sufficient to control
secretion of the target protein by the yeast cells. Only the use of
a construct in which an additional, co-secreting protein component
(the mature pheromone) is processably located upstream of the
target protein to be secreted enables said target protein to be
secreted into the culture medium in the desired manner.
DETAILED DESCRIPTION OF THE INVENTION
a) General Information
[0016] The protein sequences are usually indicated in the
"one-letter code" in the description and the figures.
[0017] A protein which is intracellularly expressed by a host cell,
in particular by yeasts, and which is secreted from the cell via
endogenous cell mechanisms through the cell membrane, preferably
into the surrounding medium is "secretable" in accordance with the
present invention.
[0018] A protein precursor (i.e. a protein in its originally
expressed form, such as, for example a pre-protein, with N- and/or
C-terminal peptide sequences no longer present in the mature
processed protein) is "processable" in accordance with the present
invention, if it can be converted to the mature form by proteolytic
processes inside and/or outside the host cell.
[0019] A "processable linkage" is present if individual protein
sections in a protein to be processed are linked via peptide bonds
which can be cleaved by a proteolytic enzyme of the host cell.
[0020] "Processing" may take place N-terminally and, where
appropriate, also C-terminally of the sequence of mature, processed
protein (target protein).
[0021] Although a "homologous" target protein is originally
expressed in the host used according to the invention and is thus a
protein endogenous to the host, it is secreted by the host cells,
owing to transformation of said host with an expression construct
of the invention.
[0022] A "heterologous" target protein is originally not expressed
in the host used according to the invention and is thus not an
endogenous host protein, but is secreted by the host cells, owing
to transformation of said host with the expression construct of the
invention.
[0023] A "shuttle peptide" is part of a "shuttle peptide construct"
processable in the host cell used according to the invention. It
forms said shuttle peptide construct together with one or more
processable regulatory peptide fragments C- and/or N-terminally,
preferably N-terminally, linked therewith, such as signal
sequences, leader sequences. In contrast to the signal peptide, for
example, the shuttle peptide is a polypeptide secreted by the host
cell. Processing of the regulatory elements is preferably carried
out intracellularly. The shuttle peptide remains secretable, even
when it is, preferably C-terminally, processably fused to a target
protein. This C-terminal processing, i.e. removal of the target
protein proteolytically in the course of secretion, is preferably
carried out, for example, during passage through the envelope of
the host cell, or in the extracellular space, for example in the
surrounding culture medium, by endogenous cellular proteases.
[0024] An "expression construct" or an "expression cassette"
according to the present invention comprises, operatively linked to
the coding nucleic acid sequence of a processable shuttle peptide
construct as defined above, the start and termination signals for
transcription and, where appropriate, translation, which are
required for controlling expression in a special host system such
as, in particular, yeast cells. The expression construct in
particular encompasses binding sites for transcription factors.
There is comprised a constitutive or inducible, native or
heterologous, natural or synthetic promoter operable in the host
cell 5' upstream of the coding sequence. The expression construct
moreover encompasses a number of restriction enzyme cleavage sites
such as, for example, those for inserting said construct into an
expression vector. In addition, the expression construct may
encompass a selectable marker gene.
[0025] An "expression vector" describes a construct obtainable by
introducing an expression cassette of the invention into a
replicon, for example into a plasmid, cosmid or virus. A vector of
this kind is capable of autonomous replication or of integrating
into the host genome and comprises the required control sequences
for controlling transcription and, where appropriate, translation
of the nucleic acid sequences coding according to the invention for
a processable shuttle peptide construct as defined above.
b) Preferred Embodiments
[0026] The invention firstly relates to an expression construct
which comprises the nucleic acid sequence coding for a shuttle
peptide construct which is processable by yeast cells, has the
formula (Sig-SP), and comprises in 5'-3' orientation the nucleic
acid sequences coding for [0027] a) a signal peptide (Sig)
processably linked to [0028] b) at least one shuttle peptide (SP)
secretable by said yeast cells; and, where appropriate, one or more
nucleic acid sequences promoting processing and/or secretion and
located 5'- or 3'-terminally of the coding signal peptide
sequence.
[0029] The sequences coding for SP and Sig are within the same
reading frame and, moreover, a processable sequence between the C
terminus of Sig and the N terminus of SP is formed during
translation. An example of said processable sequence is an
artificially introduced, proteolytically cleavable natural or
synthetic adaptor sequence which is, however, preferably part of
the C terminus of Sig or the N terminus of SP. The adaptor sequence
may be processed in such a way that the cleaved sequence may be
found entirely or partially at the C terminus of Sig or the N
terminus of SP. The latter is possible as long as this has no
substantial adverse effect on, and in particular does not prevent,
the secretability of SP.
[0030] The invention in particular relates to those expression
constructs which code for a processable shuttle peptide construct
derived from a polypeptide processed by yeasts in the broadest
sense. Said yeasts are in particular those selected from among
ascomycetes. Preference is given to yeasts selected from those of
the class of Archiascomycetes, the order of
Schizosaccharomycetales, and yeasts are particularly preferably
selected from among those of the genus Schizosaccharomyces, such as
S. pombe. Although there are data which indicate that Minus cells
also secrete P factor, preference is given to using the strain
matching the mating factor (pheromone) (i.e. Plus cells for the
Plus factor (P factor) and Minus cells for the Minus factor (M
factor).
[0031] The processable shuttle peptide construct is derived, in
particular, from a pheromone pre-protein of a yeast, said pheromone
being produced from said pre-protein by N- and C-terminal
processing. The pheromone preferably has N-terminally a polypeptide
removable by processing, which encompasses in particular the
elements required for processing and/or secreting the pre-protein,
such as signal peptide and, where appropriate, leader peptide, and
also the required protease cleavage sites.
[0032] Fungal pheromones are known and have been described, for
example, both for basidiomycetes such as Ustilago maydis (Urban,
M., Kahmann, R. and Bolker, M. (1996) The biallelic a mating type
locus of Ustilago maydis: remnants of an additional pheromone gene
indicate evolution from a multiallelic ancestor (Mol Gen Genet
250(4):414-420)) or Coprinopsis cinera (Halsall, J. R., Milner, M.
J. and Casselton, L. A. (2000) Three subfamilies of pheromone and
receptor genes generate multiple B mating specificities in the
mushroom Coprineus cinereus (Genetics 154(3):1115-1123)) and for
ascomycetes such as Schizosaccharomycespombe (Imai, Y. and
Yamamoto, M. (1995) The fission yeast mating pheromone P-factor:
its molecular structure, gene structure, and ability to induce gene
expression and G1 arrest in the mating partner (Genes Dev
8(3):328-338), Davey, J. (1992) Mating pheromones of the fission
yeast Schizosaccharomyces pombe: purification and structural
characterization of M-factor and isolation and analysis of two
genes encoding the pheromone (EMBO J 11(3):951-960)), Saccharomyces
cerevisiae (Michaelis, S. and Herskowitz, I. (1988) The a-factor
pheromone of Saccharomyces cerevisiae is essential for mating (Mol
Cell Biol 8(3):1309-1318), Kurjan, J. and Herskowitz, I. (1982)
Structure of a yeast pheromone gene (MF-alpha): a putative
alpha-factor precursor contains four tandem copies of mature
alpha-factor (Cell 30(3):933-943)), Kluyveromyces delphensis (Wong,
S., Fares, M. A., Zimmermann, W., Butler, G. and Wolfe, K. H.
(2003) Evidence from comparative genomics for a complete sexual
cycle in the `asexual` pathogenic yeast Candida glabrata (Genome
Biol 4(2)R10)) and Saccharomyces kluyveri (Egel-Mitani, M. and
Hansen, M. T. (1987) Nucleotide sequence of the gene encoding the
Saccharomyces kluyveri alpha mating pheromone (Nucleic Acids Res
15(15)6303)).
[0033] Pheromones suitable according to the invention are
relatively small peptides (such as, for example, from 5 to 40 or
from 8 to 30 amino acids). Their primary sequence usually exhibits
no significant homology. They are formed as pre-proteins,
proteolytically processed and released into the culture medium.
[0034] Examples of particularly suitable pheromones or
corresponding pre-proteins are the "P factor" and "M factor" and
their pre-proteins from S. pombe. (cf. Imai, Y. and Yamamoto, M.
(1995) The fission yeast mating pheromone P-factor: its molecular
structure, gene structure, and ability to induce gene expression
and G1 arrest in the mating partner (Genes Dev 8(3):328-338),
Davey, J. (1992) Mating pheromones of the fission yeast
Schizosaccharomyces pombe: purification and structural
characterization of M-factor and isolation and analysis of two
genes encoding the pheromone (EMBO J 11(3):951-960), Kjaerulff, S.,
Davey, J. and Nielsen, O. (1994) Analysis of the structural genes
encoding M-factor in the fission yeast Schizosaccharomyces pombe:
identification of a third gene, mfm3 (Mol Cell Biol
14(6)3895-3905)).
[0035] The P-factor pre-protein, for example, has a DNA sequence
according to SEQ ID NO: 9 and a protein sequence according to SEQ
ID NO:10. The pre-protein encompasses an N-terminal signal peptide
sequence bridged with four consecutive pheromone peptide sequences
separable by processing (cf. FIG. 3).
[0036] In preferred constructs of the invention, the processable
shuttle peptide construct is designed so as to comprise a signal
polypeptide (Sig) which is processably linked to the N-terminal end
of a C-terminally processable pheromone polypeptide (Pher).
[0037] The signal polypeptide encompasses in particular the
proteolytically removable native signal polypeptide (e.g. SEQ ID
NO:4 encoded by SEQ ID NO:3) of the pheromone pre-protein or is
identical thereto.
[0038] Preference is furthermore given to the C-terminally
processed pheromone polypeptide encompassing a C-terminal protease
cleavage site.
[0039] The expression construct preferably furthermore encompasses
the nucleic acid sequence coding for a homologous or heterologous
target protein (Targ) processably linked to the C terminus of the
shuttle peptide construct (Sig-SP).
[0040] The invention preferably relates to expression constructs of
the abovementioned type, encompassing the nucleic acid sequence
coding for a fusion protein which is processable by yeast cells and
has the formula Sig-L1.sub.n-Pher-L2.sub.m-Targ in which [0041]
Sig, Pher and Targ are as defined above, [0042] L1 and L2 are
processable linkers or adaptor sequences and [0043] n and m are
independently of one another 0 or 1. Preferably, however, n is 1
and m is 0.
[0044] L1 and L2 may be natural or synthetic linkers. They
encompass at least one proteolytic processable peptide sequence.
Where appropriate, other effector functions promoting, for example,
processing, secretion, transcription and/or translation may be
associated with L1 and/or L2.
[0045] Particular preference is given to expression constructs
wherein the nucleic acid sequence coding for the processable
shuttle peptide construct encompasses a signal polypeptide
(Sig)-encoding sequence according to SEQ ID NO: 3 or a functional
equivalent thereof, operatively linked to the nucleic acid sequence
according to SEQ ID NO:5 coding for the mature P-factor pheromone
(Pher), or a functional equivalent thereof.
[0046] Preferably, the linker L2 is not present. In contrast,
preference is given to providing the linker L1 which encompasses
the sequence coding for a polypeptide according to amino acid
residues 21 to 30 in SEQ ID NO:10. L1 here bridges the signal
polypeptide with the first pheromone building block (positions 31
to 57 in SEQ ID NO:10) of the prehormone. The C-terminal end of L1
corresponds to a recognition sequence of the protease required for
proteolytic processing.
[0047] In a particularly preferred embodiment, the nucleic acid
sequence coding for the processable shuttle peptide construct
encompasses a sequence according to SEQ ID NO:1.
[0048] The same procedure may in principle also be used with the
aid of the M factor, the second pheromone present in S. pombe, and
be applied to expressing any homologous and heterologous target
proteins. There are three genomic genes (mfm1.sup.+, SEQ ID NO:42;
mfm2.sup.+, SEQ ID NO: 45; and mfm3.sup.+, SEQ ID NO:48) which in
each case encode the M factor, the pheromone of cells having the
Minus mating type. First a preprotein (SEQ ID NO: 43, 46 and 49) is
produced from each gene, which is processed in the course of
secretion. Finally, the M factor (YTPKVPYMC; SEQ ID NO:51) encoded
by SEQ ID NO: 44, 47 and 50, respectively) is released as mature
pheromone into the medium (cf. FIG. 9).
[0049] Further shuttle peptide constructs suitable according to the
invention might therefore be derived from the coding sequences
according to SEQ ID NO:42, 45 or 48 which code for M-factor signal
peptide functionally linked to an M-factor pheromone. Nonlimiting
examples of corresponding coding shuttle peptide sequences
encompass, for example, nucleotide residues 1 to 117 according to
SEQ ID NO:42; nucleotide residues 1 to 123 according to SEQ ID
NO:45; or nucleotide residues 1 to 114 according to SEQ ID NO:48;
or functionally equivalent constructs derived therefrom which
control secretion and processing of the M-factor pheromone and of a
homologous or heterologous target protein C-terminally and
proteolytically removably linked to said pheromone. Functional
equivalents may comprise the sequence sections located 5' upstream
of the coding sequence of the mature M factor (SEQ ID NO:44, 47 or
50) in an unchanged or modified (e.g. by deletion of single or
multiple nucleic acid residues) form and thus code for a shuttle
peptide which has an altered amino acid sequence and which
functionally links the mature M-factor peptide sequence to a, for
example C-terminally truncated, signal sequence section.
[0050] A target protein (Targ) expressed according to the invention
may be derived from any prokaryotic or eukaryotic organism, in
particular humans, animals or yeasts, as long as it can be
expressed, secreted and processed by the host cell in the manner
according to the invention as part of a fusion protein with the
shuttle peptide (SP). The secreted and processed product may be
therapeutically useful or may have other advantageous applicable
properties. Examples of therapeutically useful proteins which may
be mentioned here are immunoglobulins, peptide hormones, growth
factors, lymphokines, protease inhibitors and the like. Examples of
target proteins having other properties with interesting
application, which may be mentioned here, are in particular
hydrophobins.
[0051] In a particularly preferred embodiment of the invention, the
target protein is a hydrophobin, in particular a class I
hydrophobin.
[0052] Typical hydrophobins are relatively small (100.+-.25 amino
acids) moderately hydrophobic proteins having a conserved motif of
8 cysteins
(X.sub.2-38-C-X.sub.5-9-C-C-X.sub.11-39-C-X.sub.8-23-C-X.sub.5-9-C-C-X.su-
b.6-18-C-X.sub.2-13). Hydrophobins may assemble at
hydrophilic-hydrophobic interfaces to give protein films. Such
aggregates of class I hydrophobins are insoluble in SDS, while
aggregates of class II hydrophobins are soluble in SDS (Wessels, J.
G. H. (1997) Hydrophobins: Proteins that change the nature of the
fungal surface. Adv Microb Physiol 38:1-45).
[0053] Hydrophobins usable according to the invention are in
particular derived from fungi, for example from ascomycetes such as
those of the genus Aspergillus, in particular A. nidulans.
[0054] Usable hydrophobins are also known from the prior art
mentioned above and are not limited to those from fungi.
[0055] Nonlimiting examples of usable hydrophobins are selected
from among SEQ ID NO: 14 (DewA), SEQ ID NO:19 (RdlA) SEQ ID NO:20
(RdlB) SEQ ID NO:21 (HYP1) SEQ ID NO:22 (HYP4) and SEQ ID NO:56
(RodA). TABLE-US-00001 p52750 (DewA) MRFIVSLLAF TAAATATALP
ASAAKNAKLA TSAAFAKQAE GTTCNVGSIA CCNSPAETNN DSLLSGLLGA GLLNGLSGNT
GSACAKASLI DQLGLLALVD HTEEGPVCKN IVACCPEGTT NCVAVDNAGA GTKAE q91190
(RdIA) MLKKAMVAAA AAASVIGMSA AAAPQALAIG DDNGPAVANG NGAESAFGNS
ATKGDMSPQLSLVEGTLNKP CLGVEDVNVA VINLVPIQDI NVLADDLNQQ CADNSTQAKR
DGALSHVLED LSVLSANGEG R q934f8 (RdIB) MIKKVVAYAA IAASVMGASA
AAAPQAMAIG DDSGPVSANG NGASQYFGNS MTTGNMSPQM ALIQGSFNKP CIAVSDIPVS
VIGLVPIQDL NVLGDDMNQQ CAENSTQAKR DGALAHLLED VSILSSNGEG GKG
HYP1_AGABI (P49072) MISRVLVAAL VALPALVTAT PAPGKPKASS QCDVGEIHCC
DTQQTPDHTS AAASGLLGVP INLGAFLGFD CTPISVLGVG GNNCAAQPVC CTGNQFTALI
NALDCSPVNV NL HYP4_AGABI (043122) MVSTFITVAK TLLVALLFVN INIVVGTATT
GKHCSTGPIE CCKQVMDSKS PQATELLTKN GLGLGVLAGV KGLVGANCSP ITAIGIGSGS
QCSGQTVCCQ NNNFNGVVAI CTPINANV RodA LPPAHDSQFA GNGVGNKGNS
NVKFPVPENV TVKQASDKCG DQAQLSCCNK ATYAGDTTTV DEGLLSGALS GLIGAGSGAE
GLGLFDQCSK LDVAVLIGIQ DLVNQKCKQN IACCQNSPSS ADGNLIGVGL
PCVALGSIL
[0056] The RodA protein is, together with the DewA protein, part of
the outer spore capsule of A. nidulans.
[0057] The invention moreover relates to expression vectors which
encompass an expression construct as defined above in operative
linkage to at least one regulatory nucleic acid sequence.
[0058] The invention also relates to recombinant microorganisms
comprising at least one expression vector or an expression
construct as defined above, where appropriate stably integrated
into the host genome.
[0059] A "recombinant microorganism in accordance with the present
invention encompasses at least one expression vector of the
invention or an expression construct of the invention and is
derived from yeasts in the broadest sense. Said yeasts are in
particular derived from ascomycetes. Preferred yeasts are selected
from the class of Archiascomycetes, the order
Schizosaccharomycetales, and are particularly preferably selected
from among yeasts of the genus Schizosaccharomyces such as S.
pombe.
[0060] The invention further relates to shuttle peptide constructs
which can be processed by yeast cells and are derived from a yeast
pheromone pre-protein, the pheromone being derivable from said
pre-protein by N- and C-terminal processing and being
secretable.
[0061] Preference is given to those shuttle peptide constructs
which comprise a signal polypeptide N-terminally processably linked
to the C-terminally processed pheromone polypeptide.
[0062] Said signal polypeptide is preferably the proteolytically
removable native signal polypeptide of the pheromone pre-protein,
and the C-terminally processed pheromone polypeptide encompasses
the C-terminal protease cleavage site.
[0063] Preferred shuttle peptide constructs are derived here from
pheromone pre-proteins of yeasts, in particular pre-proteins of the
S. pombe factors P and M. Particularly preferred shuttle peptides
encompass an amino acid sequence according to SEQ ID NO:2 or a
functional equivalent thereof.
[0064] The invention further relates to a method for recombinant
preparation of a target protein, which comprises culturing a
recombinant microorganism as defined above, expressing the nucleic
acid sequence encoding said target protein and isolating the target
protein secreted into the culture medium, such as, for example, a
hydrophobin as defined above.
[0065] The invention furthermore relates to nucleic acids coding
for a shuttle peptide construct as defined above; and to nucleic
acids coding for a fusion protein as defined above which can be
processed and by yeast cells and encompasses a target protein.
[0066] The invention also relates to hydrophobins obtainable by a
method of the invention.
[0067] Finally, the invention relates to the use of such a
hydrophobin for surface treatment, which comprises treating in
particular the surface of objects selected from among glass,
fibers, fabrics, leather, painted objects such as, for example,
motor vehicle bodies, films, facades.
[0068] The invention also relates to the use of hydrophobins for
treating the surfaces of fibers, fabrics and leather.
c) Further Embodiments of the Invention
c1) Polypeptides/Proteins
[0069] The invention also comprises "functional equivalents" of the
specifically disclosed or used polypeptide/proteins. This applies
both to the intermediately produced fusion proteins and to
components thereof, i.e. target proteins (Targ), shuttle peptides
(SP), such as pheromones (Pher), but also to signal peptides (Sig)
and linkers. The generic term used for polypeptide/protein will
only be "polypeptide" hereinbelow.
[0070] "Functional equivalents" or analogs of the specifically
disclosed polypeptides are, within the scope of the present
invention, polypeptides differing therefrom which furthermore have
the desired biological activity. Analogous shuttle peptides should
furthermore be suitable for controlling secretion and processing of
the target protein. Correspondingly, the functional equivalents of
components of the shuttle peptide, such as signal polypeptide,
pheromone, linker, are also intended to have furthermore the
properties required for an effective secretion and processing of
the fusion protein with release of the target protein.
[0071] "Functional equivalents" of inventive polypeptides, such as
target proteins, shuttle peptides, may comprise at the C and/or N
terminals in particular remnants of natural linker or adaptor
sequences, which result from proteolytic cleavage.
[0072] "Functional equivalents" mean according to the invention in
particular mutant proteins which have in at least one of the
sequence positions of the abovementioned specific sequences an
amino acid different from the one specifically mentioned, but which
have nevertheless one of the abovementioned biological activities.
"Functional equivalents" thus encompass the mutant proteins
obtainable by one or more amino acid additions, substitutions (cf.
examples in the table below), deletions and/or inversions, it being
possible for said modifications to occur in any sequence position,
as long as they result in a mutant protein having the property
profile of the invention.
[0073] Examples of residues suitable for amino acid substitutions
are: TABLE-US-00002 Original residue Substitution examples Ala Ser
Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His
Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile
Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile;
Leu
[0074] There is functional equivalence in particular also when the
mutant and the unaltered polypeptide have qualitatively matching
activity patterns. This means, for example, that modified shuttle
peptides express or secrete the same target protein in the same
host with higher or lower efficiency; or that modified target
proteins have an increased or reduced pharmacological action or
modified applicable property.
[0075] "Functional equivalents" in the above sense also encompass
precursors of the polypeptides described and also functional
derivatives and salts of said polypeptides. The expression "salts"
means both salts of carboxyl groups and acid addition salts of
amino groups of the protein molecules of the invention. Salts of
carboxyl groups may be prepared in a manner known per se and
encompass inorganic salts such as, for example, sodium, calcium,
ammonium, iron and zinc salts and also salts with organic bases
such as, for example, amines, such as triethanolamine, arginine,
lysine, piperidine and the like. The invention likewise relates to
acid addition salts such as, for example, salts with mineral acids
such as hydrochloric acid or sulfuric acid and salts with organic
acids such as acetic acid and oxalic acid.
[0076] "Functional derivatives" of polypeptides of the invention
may likewise be prepared on functional amino acid side groups or on
their N- or C-terminal ends with the aid of known techniques.
Derivatives of this kind encompass, for example, aliphatic esters
of carboxylic acid groups, amides of carboxylic acid groups,
obtainable by reaction with ammonia or with a primary or secondary
amine; N-acyl derivatives of free amino groups, prepared by
reaction with acyl groups; or O-acyl derivatives of free hydroxy
groups, prepared by reaction with acyl groups.
[0077] "Functional equivalents" also encompass, of course,
polypeptides obtainable from organisms different from those
specifically mentioned and also naturally occuring variants. For
example, it is possible to establish by sequence comparison regions
of homologous sequence regions and to determine equivalent enzymes
following the specific guidelines of the invention.
[0078] "Functional equivalents" also encompass fragments,
preferably individual domains or sequence motifs, of the
polypeptides of the invention, which have the desired biological
function, for example.
[0079] "Functional equivalents" are moreover fusion proteins which
have one of the above-mentioned polypeptide sequences or functional
equivalents derived therefrom and at least one further heterologous
sequence functionally different therefrom in functional N- or
C-terminal linkage (i.e. with mutual negligible functional
impairment of the fusion protein parts). Nonlimiting examples of
heterologous sequences of this kind are, for example, signal
peptides, enzymes, immunoglobulins, surface antigens, receptors or
receptor ligands.
[0080] "Functional equivalents" also encompassed by the invention
are homologs of the specifically mentioned polypeptides. Said
homologs are at least 60%, preferably at least 75%, in particular
at least 85%, such as, for example, 90%, 95% or 99%, homologous to
any of the specifically disclosed sequences, calculated according
to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci.
(USA) 85(8), 1988, 2444-2448. A percentage homology of a homologous
polypeptide of the invention means in particular percentage
identity of the amino acid residues based on the total length of
any of the amino acid sequences specifically described herein.
[0081] In the case of a possible protein glycosylation, equivalents
of the invention encompass polypeptides in deglycosylated or
glycosylated form and modified forms obtainable by altering the
glycosylation pattern.
[0082] Homologs of the proteins or polypeptides of the invention
may be generated in a manner known per se by mutagenesis, for
example by point mutagenesis or truncation of the protein.
c2) Nucleic Acid Sequences:
[0083] Nucleic acid sequences of the invention, in particular those
coding for any of the above polypeptides and their functional
equivalents, encompass single- and double-stranded DNA and RNA
sequences such as, for example, also cDNA and mRNA.
[0084] All nucleic acid sequences mentioned herein either are of
natural origin or can be prepared in a manner known per se by
chemical synthesis of nucleotide building blocks such as, for
example, by fragment condensation of individual overlapping,
complementary nucleic acid building blocks.
[0085] The chemical synthesis of oligonucleotides may be carried
out in a manner known per se, for example according to the
phosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New
York, pages 896-897). The assembly of synthetic oligonucleotides
and filling-in of gaps with the aid of the Klenow fragment of DNA
polymerase and ligation reactions and also general cloning methods
are described in Sambrook et al. (1989), Molecular Cloning: A
laboratory manual, Cold Spring Harbor Laboratory Press.
[0086] The invention also relates to nucleic acid sequences coding
for any of the above polypeptides and their functional equivalents,
which are accessible by using artificial nucleotide analogs, for
example.
[0087] The invention relates both to isolated nucleic acid
molecules coding for polypeptides of the invention or biologically
active sections thereof and to nucleic acid fragments suitable, for
example, for use as hybridization probes or primers for identifying
or amplifying coding nucleic acids of the invention.
[0088] The nucleic acid molecules of the invention may moreover
comprise untranslated sequences of the 3' and/or 5' end of the
coding gene region.
[0089] An "isolated" nucleic acid molecule is removed from other
nucleic acid molecules present in the natural source of said
nucleic acid and may, in addition, be essentially free of other
cellular material or culture medium when prepared by recombinant
techniques, or free of chemical precursors or other chemicals, when
synthesized chemically.
[0090] A nucleic acid molecule of the invention may be isolated by
means of molecular-biological standard techniques and the sequence
information provided according to the invention. For example, cDNA
may be isolated from a suitable cDNA bank by using any of the
specifically disclosed complete sequences or a section thereof as
hybridization probe and standard hybridization techniques (as
described, for example, in Sambrook, J., Fritsch, E. F. and
Maniatis, T. Molecular Cloning: A Laboratory Manual. 2.sup.nd
edition, Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989). In addition, it
is possible to isolate a nucleic acid molecule encompassing any of
the disclosed sequences or a section thereof by polymerase chain
reaction, using the oligonucleotide primers produced on the basis
of said sequence. The nucleic acid amplified in this way may be
cloned into a suitable vector and characterized by DNA sequence
analysis.
[0091] The invention furthermore encompasses the nucleic acid
molecules complementary to the specifically described nucleotide
sequences or a section thereof.
[0092] Said nucleotide sequences enable probes and primers to be
generated which may be used for identifying and/or cloning
homologous sequences in other cell types and organisms. Such probes
or primers usually encompass a nucleotide sequence region which
hybridizes under stringent conditions to at least about 12,
preferably to at least about 25, such as, for example, about 40, 50
or 75, consecutive nucleotides of a sense strand of a nucleic acid
sequence of the invention or of a corresponding antisense
strand.
[0093] Further nucleic acid sequences of the invention are derived
from the specifically disclosed sequences and differ therefrom by
addition, substitution, insertion or deletion of single or multiple
nucleotides, but still code for polypeptides having the desired
property profile.
[0094] The invention also encompasses those nucleic acid sequences
which encompass "silent" mutations or which have been modified
according to the codon usage of a special source or host organism,
in comparison with a specifically mentioned sequence, as well as
naturally occurring variants thereof such as, for example, splice
variants or allele variants. The invention also relates to
sequences obtainable by conservative nucleotide substitutions
(replacing the amino acid in question with an amino acid of
identical charge, size, polarity and/or solubility).
[0095] The invention also relates to the molecules derived from the
specifically disclosed nucleic acids by sequence polymorphisms.
These genetic polymorphisms may exist between individuals within a
population due to natural variation. These natural variations
usually cause a variance of from 1 to 5% in the nucleotide sequence
of a gene.
[0096] The invention furthermore also encompasses nucleic acid
sequences which hybridize with abovementioned coding sequences or
which are complementary thereto. These polynucleotides can be found
when screening genomic or cDNA banks and, where appropriate, be
amplified therefrom by means of PCR using suitable primers and
subsequently be isolated using suitable probes, for example.
[0097] The property of being able to "hybridize" to polynucleotides
means the ability of a poly- or oligonucleotide to bind under
stringent conditions to a nearly complementary sequence, while
unspecific bondings between noncomplementary partners do not occur
under these conditions. For this purpose, the sequences should be
70-100%, preferably 90-100%, complementary. The property of
complementary sequences of being able to specifically bind to one
another is utilized, for example, in the Northern or Southern blot
technique or for primer binding in PCR or RT-PCR. Usually
oligonucleotides of 30 or more base pairs in length are used for
this purpose. Stringent conditions mean, for example in the
Northern blot technique, the use of a washing solution at
50-70.degree. C., preferably 60-65.degree. C., for example
0.1.times.SSC buffer containing 0.1% SDS (20.times.SSC: 3M NaCl,
0.3M sodium citrate, pH 7.0) for eluting unspecifically hybridized
cDNA probes or oligonucleotides. This means, as mentioned above,
only highly complementary nucleic acids remain bound to one
another. Setting stringent conditions is known to the skilled
worker and is described, for example, in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6.
c3) Expression Constructs and Vectors:
[0098] The invention moreover relates to expression constructs
comprising a nucleic acid sequence coding for a polypeptide to be
expressed according to the invention under the genetic control of
regulatory nucleic acid sequences; and to vectors encompassing at
least one of said expression constructs.
[0099] Such constructs of the invention preferably encompass a
promoter 5' upstream of the particular coding sequence and a
terminator sequence 3' downstream and, where appropriate, further
common regulatory elements, in each case operatively linked to the
coding sequence.
[0100] "Operative linkage" means the sequential arrangement of
promoter, coding sequence, terminator and, where appropriate, other
regulatory elements such that each of the regulatory elements can
fulfill, as required, its function in expressing the coding
sequence.
[0101] Examples of operatively linkable sequences are targeting
sequences and also enhancers, polyadenylation signals and the like.
Other regulatory elements encompass selectable markers,
amplification signals, origins of replication and the like.
Suitable regulatory sequences are described, for example, in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990).
[0102] The gene construct may comprise one or more copies of the
coding nucleic acid sequences.
[0103] Examples of usable promoters are the yeast promoters ADC1,
MFalpha , AC, P-60, CYC5, GAPDH, nmt1, nmt41 and nmt81.
[0104] Examples of suitable promoters for the yeast S. pombe, which
may be mentioned, are: nmt1, nmt41, nmt81, adh1, fbp1, SV40 or
CaMV. Further information under
(http://pingu.salk.edu/.about.forsburg/vectors.html#exp). The
promoters differ with respect to their rate of transcription. The
selection depends on the desired level of expression. This applies
to other yeasts accordingly.
[0105] Suitable yeast promoters are described, for example, in the
published US patent application 2003/0077831 to which reference is
expressly made hereby.
[0106] It is also useful to use inducible promoters such as, for
example, light- and, in particular, temperature-inducible
promoters.
[0107] The regulatory sequences mentioned are intended to make
possible targeted expression of said nucleic acid sequences and of
protein expression. Depending on the host organism, this may mean,
for example, that the gene is expressed or overexpressed only after
induction or that it is expressed and/or overexpressed
immediately.
[0108] In this connection, the regulatory sequences or factors may
preferably influence expression in a positive way and thereby
increase or reduce the latter. Thus, the regulatory elements may
advantageously be enhanced at the transcriptional level by using
strong transcription signals such as promoters and/or "enhancers".
Apart from this, however, it is also possible to enhance
translation by improving, for example, mRNA stability.
[0109] An expression cassette is prepared by fusing a suitable
promoter to a suitable coding nucleotide sequence and to a
terminator signal or polyadenylation signal. For this purpose,
common recombination and cloning techniques are used, such as those
described, for example, in T. Maniatis, E. F. Fritsch and J.
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) and in T. J.
Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene
Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1984) and in Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Greene Publishing Assoc. and Wiley Interscience
(1987).
[0110] The recombinant nucleic acid construct or gene construct is
expressed in a suitable host organism by inserting it
advantageously into a host-specific vector which makes optimal
expression of the genes in the host possible. Vectors are well
known to the skilled worker and can be found, for example, in
"Cloning Vectors" (Pouwels P. H. et al., eds., Elsevier,
Amsterdam-New York-Oxford, 1985). Vectors mean, apart from
plasmids, also any other vectors known to the skilled worker, such
as, for example, phages, viruses, such as SV40, CMV, baculovirus
and adenovirus, transposons, IS elements, phasmids, cosmids and
linear or circular DNA. These vectors may be replicated
autonomously in the host organism or chromosomally.
[0111] Examples of expression vectors suitable according to the
invention, which may in particular be mentioned here, are
constructs suitable for the yeast S. pombe (see e.g.:
(http://pingu.salk.edu/.about.forsburg/vectors.html#exp).
Other Examples are:
[0112] pART1 (McLeod, M., Stein, M., and Beach, D. (1987) The
product of the mei3+ gene expressed under control of the mating
type locus, induces meiosis and sporulation in fission yeast. EMBO
J. 6:729-736
[0113] pCHY21 (Hoffman, C. S. and Winston, F. (1991). Glucose
repression of transcription of the schizosaccharomyces pombe fbp1
gene occurs by a camp signaling pathway. Genes Dev. 5:561-571)
[0114] REP1 ,REP3, REP4 (Maundrell, K. (1990). nmt1 of fission
yeast: a highly transcribed gene completely repressed by thiamine.
J. Biol. Chem. 265:10857-10864)
[0115] REP41, REP42, REP81, REP82 (Basi, G., Schmid, E. and
Maundrell, K. (1993). TATA box mutations in the Schizosaccharomyces
pombe nmt1 promoter affect transcription efficiency but not the
transcription start point or thiamine repressibility. Gene
123:131-136)
[0116] Yeast expression vectors for expression in the yeast S.
cerevisiae, such as pYEpSec1 (Baldari et al., (1987) Embo J.
6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123) and pYES2
(Invitrogen Corporation, San Diego, Calif.). Vectors and methods
for constructing vectors, which are suitable for use in other fungi
such as filamentous fungi, include those which are described in
detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene
transfer systems and vector development for filamentous fungi, in:
Applied Molecular Genetics of Fungi, J. F. Peberdy et al., eds.,
pp. 1-28, Cambridge University Press: Cambridge.
[0117] Other suitable expression systems are described in chapters
16 and 17 of Sambrook, J., Fritsch, E. F. and Maniatis, T.,
Molecular cloning: A Laboratory Manual, 2.sup.nd edition, Cold
Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1989.
c4) Recombinant Microorganisms:
[0118] It is possible with the aid of the vectors of the invention,
to prepare recombinant microorganisms which have been transformed,
for example, with at least one vector of the invention and which
may be used for producing the polypeptides of the invention.
[0119] Advantageously, the above-described recombinant constructs
of the invention are introduced into a suitable host system and
expressed. Preferably, this involves using familiar cloning and
transfection methods known to the skilled worker, such as, for
example, coprecipitation, protoplast fusion, electroporation,
retroviral transfection and the like, in order to make the nucleic
acids mentioned be expressed in the particular expression system.
Suitable systems are described, for example, in Current Protocols
in Molecular Biology, F. Ausubel et al., eds., Wiley Interscience,
New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory
Manual. 2.sup.nd ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0120] Suitable host organisms are in principle any organisms which
enable nucleic acids of the invention, their allelic variants,
their functional equivalents or derivatives to be expressed.
Preferred host organisms are yeasts.
[0121] Methods for introducing exogenous DNA into yeast cells are
known from the prior art. Said introduction may be carried out, for
example, by spheroplast transformation according to Hinnen et al.
(1978, Proc. Natl. Acad. Sci., USA, 75: 1919-1935). Chemical
transformation methods can be found, for example for S. pombe, in
Alfa et al. (Alfa, C., Fantes, P., Hyams, J., McLeod, M. and
Warbrick, E. (1993) Experiments with fission yeast. Cold Spring
Harbor Laboratory Press, New York) or, for S. cerevisiae, in Kaiser
et al. (Kaiser, C., Michaelis, S. and Mitchell, A. (1994) Methods
in Yeast Genetics. Cold Spring Harbor Laboratory Press, New
York).
[0122] Auxotrophic markers are frequently utilized in yeasts to
select transformants. In this case, the strain to be transformed
lacks a protein required for producing particular metabolic
products. The corresponding active protein is introduced into the
cell by the utilized vector. Frequently utilized markers are genes
of uracil, leucine, histidine or tryptophan biosynthesis, for
example.
[0123] Successfully transformed organisms may be selected via
marker genes which are also included in the vector or in the
expression cassette. Examples of such marker genes are genes for
antibiotic resistance and for enzymes catalyzing a color-producing
reaction which causes the transformed cell to be stained. The
latter may then be selected by means of automated cell sorting.
Microorganisms which have been transformed successfully with a
vector and which carry an appropriate antibiotic resistance gene
(e.g. G418 or hygromycin) can be selected via corresponding
antibiotic-containing media or nutrient beds. Marker proteins
exposed on the cell surface may be utilized for selection by means
of affinity chromatography.
[0124] The combination of the host organisms and the appropriate
vectors for said organisms, such as plasmids, viruses or phages,
for example plasmids containing the RNA polymerase/promoter system,
the phages 8 or: or other temperate phages or transposons, and/or
other advantageous regulatory sequences constitutes an expression
system.
c5) Recombinant Preparation of Target Proteins
[0125] The invention furthermore relates to methods for recombinant
preparation of a target protein as defined above.
[0126] The recombinant microorganism may be cultured and fermented
according to known methods. Suitable culturing conditions are
described in detail, for example for S. pombe in Alfa et al. (Alfa,
C., Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993)
Experiments with fission yeast. Cold Spring Harbor Laboratory
Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U.
and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of
Genetics 1, pp 395-446, Plenum Press, New York) or, for S.
cerevisiae, in Kaiser et al. (Kaiser, C., Michaelis, S. and
Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor
Laboratory Press, New York).
[0127] If the target protein is secreted into the culture
supernatant, the cells are removed from the latter and the target
protein is obtained from the supernatant according to known protein
isolation methods.
[0128] The target protein may be purified using known,
chromatographic methods such as molecular-sieve chromatography (gel
filtration), ion exchange chromatography such as Q-Sepharose
chromatography, and hydrophobic chromatography, and using other
common methods such as ultrafiltration, crystallization, salting
out, dialysis and native gel electrophoresis. Suitable methods are
described, for example, in Cooper, F. G., Biochemische
Arbeitsmethoden [Original title: The Tools of Biochemistry], Verlag
Walter de Gruyter, Berlin, New York or in Scopes, R., Protein
Purification, Springer Verlag, New York, Heidelberg, Berlin.
[0129] Isolation of the recombinant protein may also be facilitated
by using vector systems coding for altered polypeptides or fusion
proteins which simplify purification. Examples of suitable
modifications of this kind are "Tags" acting as anchors, such as,
for example, the modification known as hexa-histidine anchor or
epitopes which can be recognised as antigens by antibodies
(described, for example, in Harlow, E. and Lane, D., 1988,
Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
These anchors may be used for attaching the proteins to a solid
support such as, for example, a polymer matrix which can, for
example, be packed into a chromatography column, or may be used on
a microtiter plate or another support.
[0130] These anchors may at the same time also be used for
recognition of the proteins. Moreover, the proteins may be
recognised by using conventional markers such as fluorescent dyes,
enzyme markers forming a detectable reaction product after reaction
with a substrate, or radioactive labels, alone or in combination
with said anchors for derivatizing the proteins.
c6) Surface Treatment with Hydrophobin
[0131] The treatment of surfaces with hydrophobins in order to
alter, for example hydrophobize or hydrophilize, the surface
properties is known in principle, and is now substantially
simplified by the invention whose recombinant preparation of the
hydrophobins provides sufficient starting material.
[0132] Taking into account the teaching of the prior art (such as,
for example, that of WO-A-01/57066 which describes stabilization,
solubilization and, related thereto, improved application of
hydrophobins due to sulfite treatment; or of WO-A-01/57076 which
describes purification of hydrophobin via adsorption to Teflon
beads and elution by means of a detergent such as Tween at low
temperatures; or of WO-A-01/57528 which describes fixing
hydrophobins to surfaces by applying Tween and temperatures up to
85 degrees Celsius), a very large variety of solid materials such
as glass, fibers, fabrics, leather, painted objects, films, facades
can be coated with hydrophobin.
[0133] The following nonlimiting examples describe the invention in
more detail with reference to the attached figures in which
[0134] FIG. 1 depicts various constructs, prepared according to the
invention, for secretion of hydrophobins from S. pombe.
[0135] FIG. 2A) depicts the genomic sequence of the DewA gene (SEQ
ID NO:39); the sequences of the two introns are underlined; B)
depicts the amino acid sequence and, in parentheses, the
corresponding DNA sequence of the Aspergillus nidulans DewA
protein; the signal sequence is printed in bold type, and the
partial sequence following the signal sequence corresponds to the
sequence of mature DewA; C) depicts the amino acid sequence and, in
parentheses, the corresponding DNA sequence of the HA-Tag.
[0136] FIG. 3A) depicts the amino acid sequence and, in
parentheses, the corresponding DNA sequence of the P-factor
pre-protein; the signal sequence is printed in bold type; the
underlined partial sequences following the signal sequence
correspond to the sequences of the four mature pheromone peptides;
the pheromone next to the signal peptide is referred to as
P-factor; B) depicts the amino acid sequence and, in parentheses,
the corresponding DNA sequence of the removable signal peptide and,
downstream therefrom, the 6 amino acids (underlined) of the
P-factor pre-protein; C) depicts the amino acid sequence and, in
parentheses, the corresponding DNA sequence of the "P-shuttle" of
the invention; the signal sequence is printed in bold type, the
underlined partial sequence following the signal sequence
corresponds to the sequence of mature P-factor;
[0137] FIG. 4 depicts a fusion protein of the invention, comprising
the "P-shuttle" sequence (signal sequence in bold; mature P protein
underlined), the mature DewA (double-underlined) and the
C-terminally fused HA-Tag (SEQ ID NO:18; encoded by SEQ ID
NO:17);
[0138] FIG. 5 depicts the immunological detection of hydrophobins
in S. pombe* For immunological detection, the A. nidulans
hydrophobin genes DewA and RodA were fused to an HA tag, cloned
into the pJR1-3XL expression vector and transformed into S. pombe.
"Membrane fraction" and "cytosoli proteins" were fractionated by
SDS-PAGE. Detection in the Western analysis was carried out using
HA antibodies. The size standard in kDa is indicated on the left.
A: samples of a culture containing the insert-free vector
(pJRI-3XL, negative control), an control) and a vector comprising
the HA-tagged DewA gene with introns (DewA-HA(+introns)) were
applied. B: samples of a culture containing the vector (pJR1-3XL,
negative control), a vector comprising the HA-tagged DewA gene
without introns (DewA-HA(-introns)) or the HA-tagged RodA gene with
introns (RodA-HA(+introns)) were applied in each case.
[0139] FIG. 6 depicts the immunological detection of the expression
of hydrophobins in S. pombe. The PDewAHA protein was expressed in
S. pombe. The cells were harvested, the culture supernatant was
divided into aliquots and a part was precipitated with TCA. The
protein was detected after SDS-PAGE and Western blot with the aid
of HA antibodies. The bands indicated by * correspond to the
precursor protein (approx. 18 kD, upper band) and the mature form
(approx. 17 kD, lower band).
[0140] FIG. 7 depicts the detection of expression of hydrophobins
in S. pombe. S. pombe cells were transformed with plasmids which
express P+6DewA via a strong promoter (pJR1-3XL) or a weaker
promoter (pJR1-81XL). The cells carry chromosomally a version of
the prp1 gene with a c-myc-Tag which serves as control in order to
rule out contamination of the culture supernatant by lysed cells.
Cells were harvested (pellet), the culture supernatant was
precipitated with TCA (supernatant). The proteins were detected
after SDS-PAGE and Western blot with the aid of antibodies against
HA (A) or against c-myc (B).
[0141] FIG. 8 depicts the detection of secretion with the aid of
the "P shuttle" method. S. pombe cells were transformed with
plasmids which express PfakDewA via a weaker promoter (pJR1-81XL).
The cells were harvested (pellet) and the culture supernatant was
precipitated with TCA (SN). The protein was detected after SDS-PAGE
and Western blot with the aid of antibodies against HA.
[0142] FIG. 9 depicts the three genes which encode in each case the
S. pombe M-factor (SEQ ID NO:51 for mature factor): A) depicts
sequences for the mfm1.sup.+- gene; B) depicts sequences for the
mfm2.sup.+- gene; and C) sequences for the mfm3.sup.+- gene.
[0143] FIG. 10 depicts the RodA gene. The genomic sequence (SEQ ID
NO:52) of the RodA gene comprises two introns (underlined) which
are not present in the corresponding coding ORF (SEQ ID NO:53). The
pre-protein (SEQ ID NO:54) comprises a removable signal sequence
(printed in bold type) which is absent in the mature protein (SEQ
ID NO:56; encoded by SEQ ID NO:55).
EXPERIMENTAL SECTION
General Experimental Details:
a) General Cloning Methods
[0144] The cloning steps carried out for the purpose of the present
invention, such as, for example, restriction cleavages, agarose gel
electrophoresis, purification of DNA fragments, transfer of nucleic
acids to nitrocellulose and nylon membranes, linking of DNA
fragments, transformation of E. coli cells, culturing of bacteria,
propagation of phages and sequence analysis of recombinant DNA were
carried out as described by Sambrook et al. (1989) loc. cit.,
unless stated otherwise.
[0145] DNA was purified from reaction mixtures or after gel
electrophoresis by means of the NucleoSpin Extract Kit
(Machery-Nagel, Duren, Germany) and plasmid DNA was isolated from
E. coli with the aid of the NucleoSpin Plasmid Quick Pure Kit
(Machery-Nagel, Duren, Germany) according to the manufacturer's
information.
[0146] Restriction enzymes (Invitrogen) were used according to the
manufacturer's information. Ligations of DNA were carried out with
the aid of T4 ligase (Promega, Mannheim, Germany) according to the
manufacturer's information.
[0147] Transformations into E. coli were carried out by means of
electroporation using the Gene Pulser II apparatus (BIO-RAD,
Munich, Germany) and 2-mm electroporation cuvettes (Biozym
Diagnostik, Hess. Oldendorf, Germany) according to the
manufacturers' information. Transformants were selected on LB
medium (Lennox, 1955, Virology, 1:190) containing ampicillin (150
mg/l).
b) Polymerase Chain Reaction (PCR)
[0148] PCR amplifications were carried out with the aid of
Combizyme DNA polymerase (Invitek, Berlin, Germany) according to
the manufacturer's information. In each case 1 pmol of the
appropriate primers was used per 100 .mu.l of reaction volume.
c) Culturing
[0149] S. pombe was cultured as described in Alfa et al. (Alfa, C.,
Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993)
Experiments with fission yeast. Cold Spring Harbor Laboratory
Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U.
and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of
Genetics 1, pp 395-446, Plenum Press, New York).
[0150] Recombinant strains were cultured as described in Alfa et
al. (Alfa, C., Fantes, P., Hyams, J., McLeod, M. and Warbrick, E.
(1993) Experiments with fission yeast. Cold Spring Harbor
Laboratory Press, New York) und Gutz et al. (Gutz, H., Heslot, H.,
Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In:
Handbook of Genetics 1, pp 395-446, Plenum Press, New York).
d) Cell Disruption
[0151] For quick controls of expression, the cells were removed by
centrifugation (5 min, 3.500.times.g) and the cell pellet was taken
up directly in Laemmli buffer (Laemmli, U.K. (1970) Cleavage of
structural proteins during the assembly of the head of
bacteriophage T4 (Nature 227:680-685)) for gel electrophoresis.
[0152] For cell disruption, the cells were harvested by
centrifugation at 3.500.times.g for 5 min. The cell pellets were
resuspended in 1 ml of 1.times.PBS and 1 volume of glass beads was
added. The mixture was vortexed for 5 min, and the supernatant
above the glass beads was removed.
e) Organisms Used
[0153] The strains DH5.alpha. (Invitrogen), XL10-Gold (Stratagene)
or BL21 (BioLabs) were used for E. coli work.
[0154] S. pombe strains were taken from the fission yeast strain
collection of the group of Prof. Dr. G. Rodel of the Institute for
Genetics of Technische Universitat Dresden, Germany.
Example 1
Preparation of the DewA and DewAHA Expression Constructs and
Cloning into Vector pJR1-3XL
a) Isolation of the Genomic DNA Sequence of the DewA Gene and
Removal of Introns
[0155] A. nidulans chromosomal DNA which had been isolated as in
Kaiser et al. (Kaiser, C., Michaelis, S. and Mitchell, A. (1994)
Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, New
York) was kindly provided by Prof. Dr. A. Brakhage (Hanover,
Germany).
[0156] The approx. 550 bp genomic DNA fragment was PCR-amplified
using chromosomal DNA as template and the primers SpDewBamrev and
ScDewBamfor.
[0157] ScDewBamfor: TABLE-US-00003 (SEQ ID NO:41) 5' - TAA TAA GGA
TCC ATG CGC TTC ATC GTC TCT CTC C - 3'
[0158] SpDewBamrev: TABLE-US-00004 (SEQ ID NO:28) 5' - TAA TAA GGA
TCC TTA CTC AGC CTT GGT ACC GGC -3'
[0159] The reaction mixture was fractionated by gel electrophoresis
and the corresponding DNA band was eluted as described above. The
fragment which is flanked on either side by a BamHl cleavage site
introduced by the primers was cleaved with the restriction
endonuclease BamHl (Invitrogen) according to the manufacturer's
information and purified from the reaction mixture (see above).
[0160] The vector pUC18 (Yanisch-Pron, C., Vieira, J. and Messing,
J. (1985) Improved M13 phage cloning vectors and host strains:
Nucleotide sequences of M13mpl8 and pUC19 vectors. Gene 33:103) was
likewise cleaved with BamHl, fractionated by gel electrophoresis
and subsequently eluted from the gel (see above).
[0161] Vector and fragment were ligated (see above) and the
ligation mixture was transformed into E. coli. Recombinant plasmids
were identified after plasmid preparation and subsequent
restriction digestion. After cloning, the correct DNA sequence of
the cloned PCR products was verified by sequencing, and this was
also done for all constructs prepared hereinbelow. Sequencing
reactions were carried out according to Sanger et al. (Sanger, F.,
Nicklen, S. and Coulson, A. R. (1977) DNA sequencing with chain
terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467). The
sequencing reactions were carried out with the aid of the
"Thermo-Sequenase fluorescent labelled primer cycle sequencing kit
with 7-deaza-dGTP" (Amersham Pharamacia Biotech, Freiburg, Germany)
and 5' IRD800-labeled primers (MWG Biotech AG, Ebersberg, Germany).
The products were fractionated and the sequence was analyzed using
the automated sequencing system LI-COR 4000/4200 (MWG Biotech AG,
Ebersberg, Germany).
[0162] A construct containing the intron-containing, genomic DewA
gene cloned into the BamHl cleavage site of the pUC18 vector was
referred to as pDewAgen.
[0163] The two introns present in the genomic DewA DNA (see genomic
DewA sequence SEQ ID NO: 39) were removed by means of "Overlap
Extension PCR" (OEP) (cf. Pogulis, R. J., Vallejo, A. N. and Pease,
L. R. In vitro recombination and mutagenesis by overlap extension
PCR. Methods Mol Biol. 1996; 57:167-76).
[0164] Using DNA of the pDewAgen construct as template,
subfragments of the open reading frame (ORF) of DewA were first
PCR-amplified with the aid of the primer pairs
ScDewBamfor/Dewl1rev, Dewl1for/Dewl2rev and Dewl2for/SpDewBamrev.
TABLE-US-00005 ScDewBamfor: (SEQ ID NO:41) 5' - TAA TAA GGA TCC ATG
CGC TTC ATC GTC TCT CTC C - 3' Dewl1rev: (SEQ ID NO:24) 5' - GT GTG
GTC GAC GAG AGC GAG CAG ACC CAG CTG - 3' Dewl1for: (SEQ ID NO:23)
5' - CAG CTG GGT CTG CTC GCT CTC GTC GAC CAC AC - 3' Dewl2rev: (SEQ
ID NO:26) 5' - GTC GAC GGC AAC ACA GTT GGT GGT TCC CTC -3'
Dewl2for: (SEQ ID NO:25) 5' - GAG GGA ACC ACC AAC TGT GTT GCC GTC
GAC -3' SpDewBamrev: (SEQ ID NO:28) 5' - TAA TAA GGA TCC TTA CTC
AGC CTT GGT ACC GGC -3'
[0165] These subfragments were fractionated by gel electrophoresis
and purified. In the final PCR, the intron-less ORF was amplified
with said subfragments as template and the distal primers
ScDewBamfor and SpDewBamrev. The approx. 410 bp PCR product was
fractionated by gel electrophoresis, purified and cleaved with the
restriction endonuclease BamHl. Appropriate cleavage sites have
been introduced by the distal primers. The cleaved fragment was
purified and cloned into the pUC 18 vector likewise cleaved with
BamHl. Vector and fragment were ligated (see above) and the
ligation mixture was transformed into E. coli. After plasmid
minipreparation, recombinant plasmids were identified and the
correct sequence of cloned ORF was verified by sequencing. The
construct obtained in this way (pDewAORF) served as template for
the construction of corresponding expression plasmids.
b) Preparation of the DewHA(+introns) and DewHA(-introns)
Expression Constructs and Introduction of a C-Terminal
Hemagglutinin Tag
[0166] Since no specific antibodies against DewA are available,
DewA was fused by OEP to the HA epitope to detect heterologous
expression. First, in the primary PCRs, the primer pairs
SpDewXhofor/DewAHArev and DewAHAfor/DewAHANcorev were utilized.
TABLE-US-00006 SpDewXhofor: (SEQ ID NO:27) 5' - TAA TAA CTC GAG ATG
CGC TTC ATC GTC TCT CTC C -3' DewAHArev: (SEQ ID NO:30) 5' - TCC
ACG CGG AAC CAG CTC AGC CTT GGT ACC -3' DewAHAfor: (SEQ ID NO:29)
5' - GGT ACC AAG GCT GAG CTG GTT CCG CGT GGA -3' DewAHANcorev: (SEQ
ID NO:31) 5' - ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC -
3'
[0167] DNA of the pDewAgen construct served as template for
preparing DewHA(+introns), and DNA of the pDewAORF construct served
as template for preparing DewHA(-introns). The vector yEP351HA
(Kettner, K., Friederichs, S., Schlapp, T. and Rodel G (2001)
Expression of a VEGF-like protein from Parapoxvirus ovis in the
yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Protein Expr Purif August 22(3):479-83) which carries the HA-tag
DNA sequence was utilized as template as template for PCR with
DewAHAfor/DewAHANcorev. The DNA coding for the HA epitope was fused
to the particular DewA-DNA in the final PCRs by means of the primer
pair SpDewXhofor/ DewAHANcorev and the respective subfragments. The
fragments amplified in this way are flanked 5' by an XhoI
restriction cleavage site and 3' by an NcoI restriction cleavage
site, both of which were introduced with the aid of the distal
primer. The fragments were fractionated by gel electrophoresis,
purified and cleaved with the restriction endonucleases XhoI and
NcoI and purified from the reaction mixture.
[0168] The vector pJR1-3XL (Moreno, M. B., Duran, A. and Ribas, J.
C. A family of multifunctional thiamine-repressible expression
vectors for fission yeast. Yeast. 2000 Jun. 30; 16(9):861-72) was
likewise cleaved with the restriction enzymes XhoI and NcoI,
fractionated by gel electrophoresis and purified. Vector and
fragment were ligated (see above) and the ligation mixture was
transformed by electroporation into E coli. After plasmid
minipreparation, recombinant plasmids were identified and the
correct sequence of the cloned ORF was verified by sequencing. In
the DewA-HA(+introns) and DewA-HA(-introns) expression plasmids
obtained in this way, expression of the fusion constructs in S.
pombe is under the control of the strong nmt1 promoter.
c) Expression of DewA-HA(+intron) and DewA-HA(-intron)
[0169] The DewA-HA(+introns) and DewA-HA(-introns) vectors obtained
according to a) and b) were transformed into the S. pombe host
strain KO103 (h.sup.-s ade6-M210 leu1-32 his7-366), as described by
Schiestl and Gietz (Schiestl, R. H. and Gietz, R. D. (1989) High
efficiency transformation of intact yeast cells using single
stranded nucleic acids as a carrier. Curr Genet 16:339-346). The
leucine auxotrophy of the S. pombe strain, caused by the leu1-32
mutation, is complemented by the S. cervisiae LEU2 gene present on
the expression vectors. It is thus possible to select transformants
on minimal medium without leucine. Expression of the fusion
proteins in corresponding yeast transformants was studied by means
of Western blot analyses.
[0170] The antibodies Anti-HA (article 1 583 816, anti-HA (12CA5)
mouse monoclonal antibody) and anti-c-myc (article 1 667 149,
anti-c-myc antibody) were purchased from Roche Diagnostics
(Mannheim, Germany).
[0171] After culturing, yeast transformants were harvested,
disrupted with glass beads, and the 3.500.times.g centrifugation
supernatant was removed. In each case 50 .mu.g of the
20.000.times.g pellets ("membrane fraction") and of the supernatant
("cytosolic proteins") were applied and fractionated in an
SDS-PAGE. Detection in the Western analysis was carried out using
HA antibodies. FIG. 5 depicts the result. The size standard in kDa
is indicated on the left. In FIG. 5A, samples of a culture
containing the insert-free vector (PJR1-3XL, negative control), an
HA-tagged control protein (positive control) or a vector comprising
the HA-tagged DewA gene with introns (DewA-HA(+introns)) were
applied. In FIG. 5B, samples of a culture containing the vector
(pJR1-3XL, negative control), or a vector comprising the HA-tagged
DewA gene without introns (DewA-HA(-Introns)) or the HA-tagged RodA
gene with introns (Rod-AHA(+introns)) were applied in each
case.
[0172] RodAHA(+introns) was prepared analogously to the information
of Example 1 a) and 1 b). RodA is another hydrophobin from A.
nidulans.
Example 2
Preparation of expression Vectors for Secretion of the Expressed
DewA Vector Comprising the PDewAHA Construct
a) Preparation of the PDewAHA Construct Comprising the Coding
Sequence for P-Factor Signal Peptide
[0173] In order to optimize secretion of the protein from S. pombe
cells, the authentic secretion signal of the A. nidulans protein,
which is not effective in fission yeast, was first replaced by the
removable signal peptide of the S. pombe P factor. The P factor is
secreted as peptide pheromone into the medium by the cells. It is
synthesized in the cell as precursor protein (pre-protein)
consisting of a removable N-terminal signal sequence and four
copies of P factor, in each case separated by short spacer
sequences, and, in the course of secretion, undergoes maturation,
including removal of the signal sequence and proteolytic release of
the four P-factor Peptides.
[0174] The P-factor signal sequence was first amplified by means of
PCR and S. pombe genomic DNA as template, using the primer pair
SigPXhofor/PDewArev, and the corresponding PCR product was
purified. TABLE-US-00007 SigPXhofor: (SEQ ID NO:34) 5' - TAA TTT
CTC GAG ATG AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA C -3'
PDewArev: (SEQ ID NO:33) 5' - GGC AGA GGC CGG GAG TGG AAT AGG TGA
GGC -3'
[0175] The PCR product of the primer pair PDewfor/DewAHANcorev and
DNA of the DewA-HA(-introns) construct as template was likewise
fractionated by gel electrophoresis and purified. TABLE-US-00008
PDewfor: (SEQ ID NO:32) 5' - GCC TCA CCT ATT CCA CTC CCG GCC TCT
GCC -3' DewAHANcorev: (SEQ ID NO:31) 5' - ATT ATT CCA TGG CTA TTA
GCG GCC GCA CTG AGC AGC -3'
[0176] These two primary PCR products were used as templates in the
final PCR with the distal primers SigPXhofor/DewAHANcorev. The
PDewAHA fragment amplified in this way is flanked 5' by an XhoI
restriction cleavage site and on its 3' end by an NcoI restriction
cleavage site, both of which were introduced with the aid of the
above primers. In the fusion protein encoded by this fragment
(PDewAHA), the removable signal sequence of A. nidulans DewA has
been replaced by the removable signal peptide of the P-factor
precursor protein.
[0177] The PDewAHA fragment was cleaved with the restriction
endonucleases XhoI and NcoI, fractionated by gel electrophoresis
and ligated into the pJR1-3XL vector (see above), which had been
cleaved with the same restriction endonucleases. Vector and
fragment were ligated (see above) and the ligation mixture was
transformed by electroporation into E. coli. After plasmid
minipreparation, recombinant plasmids were identified and the
correct sequence of the cloned ORF was verified by sequencing. The
construct obtained was referred to as PDewAHA.
b) Expression
[0178] The experiments were carried out analogously to Example
1c).
[0179] The PDewAHA protein was expressed in S. pombe. The cells
were harvested, the culture supernatant was divided into aliquots
and a part was precipitated with TCA. The TCA precipitate was taken
up in Laemmli buffer (Laemmli, U.K. (1970) Cleavage of structural
proteins during the assembly of the head of bacteriophage T4
(Nature 227:680-685)). Cell pellets, supernatant and
TCA-precipitated supernatant were studied. The protein was detected
after SDS-PAGE and Western blot with the aid of HA antibodies. FIG.
6 depicts the result. The bands indicated by * correspond to the
precursor protein (approx. 18 kD, top band) and the mature form
(approx. 17 kD, bottom band).
[0180] The analysis revealed that no effective secretion was
noticeable. The N-terminal fusion of the removable P-factor signal
peptide is not sufficient for secretion of the fused
hydrophobin.
Example 3
Preparation of Expression Vectors for Secretion of the Expressed
DewA Vector Comprising the P+6DewAHA Construct
a) Preparation of the P+6DewAHA Construct Comprising the Coding
Sequence for P-factor Signal Peptide, C-Terminally Extended by 6
Amino Acids
[0181] In order to ensure an authentic sequence environment of the
signal peptide, which may be important for secretion, the sequence
of said signal peptide in the fusion protein was extended by the 6
C-terminally adjoining amino acids (P+6DewA) by means of OEP using
the primer pair SigPXhofor/P+6DewArev and P+6DewAfor/DewAHANcorev
and DNA of the PDewAHA construct as template in the primary PCR
reactions TABLE-US-00009 SigPXhofor: (SEQ ID NO:34) 5' - TAA TTT
CTC GAG ATG AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA C -3' P +
6DewArev: (SEQ ID NO:36) 5' - CAC ACC AGG ATC GGC AAC TGG AAT AGG
TGA GGC -3' P + 6DewAfor: (SEQ ID NO:35) 5' - GTT GCC GAT CCT GGT
GTG CTC CCG GCC TCT GCC -3' DewAHANcorev: (SEQ ID NO:31) 5' - ATT
ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC -3'
and the primer pair SigPXhofor/DewAHANcorev in the final PCR
reaction.
[0182] The P+6DewA fragment was cleaved with the restriction
endonucleases XhoI and NcoI, fractionated by gel electrophoresis
and ligated into the pJR1-3XL vector (see above) which had been
cleaved with the same restriction endonucleases, and the ligation
mixture was transformed by electroporation in E. coli. After
plasmid minipreparation, recombinant plasmids were identified and
the correct sequence of the cloned ORF was verified by sequencing.
The P+6DewA fragment was also cloned into the pJR-81XL vector.
Here, transcription of the fusion gene is under the control of the
weak nmt81 promoter. This construct was intended to be used for
testing a negative influence of the very high transcription in
pJR1-3XL constructs on secretion.
[0183] The corresponding constructs were referred to as
P+6DewA/pJR1-3XL and P+6DewA/pJR1-81XL.
[0184] The experiment was carried out analogously to Example
2a.
[0185] Cloning of the amplified sequences in pJR1-3XI was carried
out analogously to Example 2a.
b) Expression
[0186] Expression was carried out analogously to Example 2b).
[0187] S. pombe cells were transformed with the two plasmids which
express P+6DewA via a strong promoter (pJR1-3XL) and a weaker
promoter (pJR1-81XL). The cells carry chromosomally a version of
the prp1 gene with a c-myc Tag which serves as a control in order
to rule out contamination of the culture supernatant by lysed
cells. The cells were harvested (pellet), and the culture
supernatant was precipitated with TCA (supernatant). The
precipitate was taken up in Laemmli buffer and likewise analyzed.
The proteins were detected after SDS-PAGE and Western blot with the
aid of antibodies against HA (FIG. 7A) and against c-myc (Roche
Diagnostics) (FIG. 7B).
[0188] As FIG. 7 illustrates, this construction is also unsuitable
for effective secretion.
Example 4
Preparation of Expression Vectors for Secretion of the Expressed
DewA Vector Comprising the PfakDewAHA Construct
a) Preparation of the PfakDewAHA Construct Comprising the Sequence
Coding for the Mature First P Factor, Including the P-Factor Signal
Peptide
[0189] DewAHA was fused by means of OEP to the carboxyl-terminal
end of the sequence of the mature P factor. The PCR fragments
obtained in the primary PCR reactions using the primer pairs
SigPXhofor/PfakDewArev and S. pombe genomic DNA as template
TABLE-US-00010 SigPXhofor: (SEQ ID NO:34) 5' - TAA TTT CTC GAG ATG
AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA C -3' PfakDewArev: (SEQ
ID NO:38) 5' - GGC AGA GGC CGG GAG GCG CTT TTT CAA GTT GGG TC -3'
and PfakDewAfor/DewAHANcorev and DNA of the P + 6DewA/pJR1-81XL
construct as template PfakDewAfor: (SEQ ID NO:37) 5' - AAC TTG AAA
AAG CGC CTC CCG GCC TCT GCC -3' DewAHANcorev: (SEQ ID NO:31) 5' -
ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC -3'
were separated by gel electrophoresis, purified and utilized as
template for the final PCR with the aid of the primer pair
SigPXhofor/ DewAHANcorev. The PfakDewA fragment obtained in this
way was cleaved with the restriction endonucleases XhoI and NcoI,
fractionated by gel electrophoresis and ligated into the pJR1-81XL
vector (see above) which had been cleaved with the same restriction
endonucleases. The ligation mixture was transformed by
electroporation into E coli. After plasmid minipreparation,
recombinant plasmids were identified and the correct sequence of
the cloned ORF was verified by sequencing. Such a construct was
referred to as PfakDewA/pJR1-81XL. In this construct, the fused
sequence coding for the P-factor pre-protein including the first
amino-terminal pheromone and for the hydrophobin is under the
control of the nmt81 promoter.
[0190] The experiment was carried out analogously to Example 2a,
but with the pJR1-81XL expression vector being used.
[0191] The amplified sequences were cloned into pJR1-81XL
analogously to Example 2a. For this purpose, the amplified DNA
which had been cleaved with the restriction endonucleases XhoI and
NcoI was cloned into the XhoI and NcoI cleavage sites of the
pJR1-81XL expression vector.
b) Expression
[0192] Expression was carried out analogously to Example 3b).
[0193] The cells were pelleted and the culture supernatant was
precipitated with TCA. The precipitate was taken up in Laemmli
buffer and likewise analyzed. The protein was detected after
SDS-PAGE and Western blot with the aid of antibodies against HA.
FIG. 8 depicts the result which shows that effective secretion of
the hydrophobin into the medium is achieved with the aid of this
construction. Thus, the corresponding fusion protein comprises all
P-factor pre-protein sequences required for secretion in their
authentic context. The P factor itself is proteolytically
removed.
Example 5
Microscopic Detection of Adsorption of Expressed Hydrophobin to
Teflon
[0194] The microscopic detection of adsorption of expressed
hydrophobin to Teflon makes use of a fluorescently labeled HA
antibody (Molecular Probes, Cat. No. A-21287).
[0195] Transformed host cells prepared according to any of Examples
1 to 4 are cultured. Cells and, where appropriate, supernatant are
harvested separately. Cells which have been transformed with a
corresponding vector without hydrophobin gene and cultured and
corresponding culture supernatants serve as reference samples.
[0196] The cells are mechanically disrupted (vibratory mill).
Teflon platelets are incubated in the cell disruption solution or
in the supernatant at room temperature for 18 h, and rinsed with
water (3.times.10 min). The treated Teflon is then incubated in PBS
with fluorescently labeled antibody, followed by rinsing again with
PBS (3.times.15 min) and drying in an N.sub.2jet. Finally, the
evaluation is carried out in a fluorescence microscope.
[0197] No fluorescence is observed on the reference sample (results
not shown), but spot-like fluorescence is obvious after incubation
in the cell homogenate or culture supernatant (when hydrophobin is
secreted by the expressing cells).
Sequence CWU 1
1
56 1 171 DNA Schizosaccharomyces pombe CDS (1)..(171) 1 atg aag atc
acc gct gtc att gcc ctt tta ttc tca ctt gct gct gcc 48 Met Lys Ile
Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10 15 tca
cct att cca gtt gcc gat cct ggt gtg gtt tca gtt agc aag tca 96 Ser
Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser 20 25
30 tat gct gat ttc ctt cgt gtt tac caa agt tgg aac act ttt gct aat
144 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe Ala Asn
35 40 45 cct gat aga ccc aac ttg aaa aag cgc 171 Pro Asp Arg Pro
Asn Leu Lys Lys Arg 50 55 2 57 PRT Schizosaccharomyces pombe 2 Met
Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10
15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser
20 25 30 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe
Ala Asn 35 40 45 Pro Asp Arg Pro Asn Leu Lys Lys Arg 50 55 3 60 DNA
Schizosaccharomyces pombe CDS (1)..(60) sig_peptide (1)..(60) 3 atg
aag atc acc gct gtc att gcc ctt tta ttc tca ctt gct gct gcc 48 Met
Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10
15 tca cct att cca 60 Ser Pro Ile Pro 20 4 20 PRT
Schizosaccharomyces pombe 4 Met Lys Ile Thr Ala Val Ile Ala Leu Leu
Phe Ser Leu Ala Ala Ala 1 5 10 15 Ser Pro Ile Pro 20 5 81 DNA
Schizosaccharomyces pombe CDS (1)..(81) 5 aag tca tat gct gat ttc
ctt cgt gtt tac caa agt tgg aac act ttt 48 Lys Ser Tyr Ala Asp Phe
Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe 1 5 10 15 gct aat cct gat
aga ccc aac ttg aaa aag cgc 81 Ala Asn Pro Asp Arg Pro Asn Leu Lys
Lys Arg 20 25 6 27 PRT Schizosaccharomyces pombe 6 Lys Ser Tyr Ala
Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe 1 5 10 15 Ala Asn
Pro Asp Arg Pro Asn Leu Lys Lys Arg 20 25 7 78 DNA
Schizosaccharomyces pombe CDS (1)..(78) sig_peptide (1)..(60) 7 atg
aag atc acc gct gtc att gcc ctt tta ttc tca ctt gct gct gcc 48 Met
Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10
15 tca cct att cca gtt gcc gat cct ggt gtg 78 Ser Pro Ile Pro Val
Ala Asp Pro Gly Val 20 25 8 26 PRT Schizosaccharomyces pombe 8 Met
Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10
15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val 20 25 9 606 DNA
Schizosaccharomyces pombe CDS (1)..(606) 9 atg aag atc acc gct gtc
att gcc ctt tta ttc tca ctt gct gct gcc 48 Met Lys Ile Thr Ala Val
Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10 15 tca cct att cca
gtt gcc gat cct ggt gtg gtt tca gtt agc aag tca 96 Ser Pro Ile Pro
Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser 20 25 30 tat gct
gat ttc ctt cgt gtt tac caa agt tgg aac act ttt gct aat 144 Tyr Ala
Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe Ala Asn 35 40 45
cct gat aga ccc aac ttg aaa aag cgc gaa ttc gaa gct gct ccc gca 192
Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe Glu Ala Ala Pro Ala 50
55 60 aaa act tat gct gat ttc ctt cgt gct tat caa agt tgg aac act
ttt 240 Lys Thr Tyr Ala Asp Phe Leu Arg Ala Tyr Gln Ser Trp Asn Thr
Phe 65 70 75 80 gtt aat cct gac aga ccc aat ttg aaa aag cgt gag ttt
gaa gct gcc 288 Val Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe
Glu Ala Ala 85 90 95 cca gag aag agt tat gct gat ttc ctt cgt gct
tac cat agt tgg aac 336 Pro Glu Lys Ser Tyr Ala Asp Phe Leu Arg Ala
Tyr His Ser Trp Asn 100 105 110 act ttt gtt aat cct gac aga ccc aac
ttg aaa aag cgc gaa ttc gaa 384 Thr Phe Val Asn Pro Asp Arg Pro Asn
Leu Lys Lys Arg Glu Phe Glu 115 120 125 gct gct ccc gca aaa act tat
gct gat ttc ctt cgt gct tac caa agt 432 Ala Ala Pro Ala Lys Thr Tyr
Ala Asp Phe Leu Arg Ala Tyr Gln Ser 130 135 140 tgg aac act ttt gtt
aat cct gac aga ccc aac ttg aaa aag cgc act 480 Trp Asn Thr Phe Val
Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Thr 145 150 155 160 gaa gaa
gat gaa gag aat gag gaa gag gat gaa gaa tac tat cgc ttt 528 Glu Glu
Asp Glu Glu Asn Glu Glu Glu Asp Glu Glu Tyr Tyr Arg Phe 165 170 175
ctt cag ttt tat atc atg act gtc cca gag aat tcc act att aca gat 576
Leu Gln Phe Tyr Ile Met Thr Val Pro Glu Asn Ser Thr Ile Thr Asp 180
185 190 gtc aat att act gcc aaa ttt gag agc taa 606 Val Asn Ile Thr
Ala Lys Phe Glu Ser 195 200 10 201 PRT Schizosaccharomyces pombe 10
Met Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5
10 15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys
Ser 20 25 30 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr
Phe Ala Asn 35 40 45 Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe
Glu Ala Ala Pro Ala 50 55 60 Lys Thr Tyr Ala Asp Phe Leu Arg Ala
Tyr Gln Ser Trp Asn Thr Phe 65 70 75 80 Val Asn Pro Asp Arg Pro Asn
Leu Lys Lys Arg Glu Phe Glu Ala Ala 85 90 95 Pro Glu Lys Ser Tyr
Ala Asp Phe Leu Arg Ala Tyr His Ser Trp Asn 100 105 110 Thr Phe Val
Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe Glu 115 120 125 Ala
Ala Pro Ala Lys Thr Tyr Ala Asp Phe Leu Arg Ala Tyr Gln Ser 130 135
140 Trp Asn Thr Phe Val Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Thr
145 150 155 160 Glu Glu Asp Glu Glu Asn Glu Glu Glu Asp Glu Glu Tyr
Tyr Arg Phe 165 170 175 Leu Gln Phe Tyr Ile Met Thr Val Pro Glu Asn
Ser Thr Ile Thr Asp 180 185 190 Val Asn Ile Thr Ala Lys Phe Glu Ser
195 200 11 156 DNA Unknown HA-tag CDS (1)..(156) 11 ctg gtt ccg cgt
gga tcc atc gaa ggt cgt ggc ggc cgc atc ttt tac 48 Leu Val Pro Arg
Gly Ser Ile Glu Gly Arg Gly Gly Arg Ile Phe Tyr 1 5 10 15 cca tac
gat gtt cct gac tat gcg ggc tat ccc tat gac gtc ccg gac 96 Pro Tyr
Asp Val Pro Asp Tyr Ala Gly Tyr Pro Tyr Asp Val Pro Asp 20 25 30
tat gca gga tcc tat cca tat gac gtt cca gat tac gct gct cag tgc 144
Tyr Ala Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Gln Cys 35
40 45 ggc cgc taa tag 156 Gly Arg 50 12 50 PRT Unknown HA-tag 12
Leu Val Pro Arg Gly Ser Ile Glu Gly Arg Gly Gly Arg Ile Phe Tyr 1 5
10 15 Pro Tyr Asp Val Pro Asp Tyr Ala Gly Tyr Pro Tyr Asp Val Pro
Asp 20 25 30 Tyr Ala Gly Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
Ala Gln Cys 35 40 45 Gly Arg 50 13 354 DNA Aspergillus nidulans CDS
(1)..(354) 13 ctc ccg gcc tct gcc gca aag aac gcg aag ctg gcc acc
tcg gcg gcc 48 Leu Pro Ala Ser Ala Ala Lys Asn Ala Lys Leu Ala Thr
Ser Ala Ala 1 5 10 15 ttc gcc aag cag gct gaa ggc acc acc tgc aat
gtc ggc tcg atc gct 96 Phe Ala Lys Gln Ala Glu Gly Thr Thr Cys Asn
Val Gly Ser Ile Ala 20 25 30 tgc tgc aac tcc ccc gct gag acc aac
aac gac agt ctg ttg agc ggt 144 Cys Cys Asn Ser Pro Ala Glu Thr Asn
Asn Asp Ser Leu Leu Ser Gly 35 40 45 ctg ctc ggt gct ggc ctt ctc
aac ggg ctc tcg ggc aac act ggc agc 192 Leu Leu Gly Ala Gly Leu Leu
Asn Gly Leu Ser Gly Asn Thr Gly Ser 50 55 60 gcc tgc gcc aag gcg
agc ttg att gac cag ctg ggt ctg ctc gct ctc 240 Ala Cys Ala Lys Ala
Ser Leu Ile Asp Gln Leu Gly Leu Leu Ala Leu 65 70 75 80 gtc gac cac
act gag gaa ggc ccc gtc tgc aag aac atc gtc gct tgc 288 Val Asp His
Thr Glu Glu Gly Pro Val Cys Lys Asn Ile Val Ala Cys 85 90 95 tgc
cct gag gga acc acc aac tgt gtt gcc gtc gac aac gct ggc gcc 336 Cys
Pro Glu Gly Thr Thr Asn Cys Val Ala Val Asp Asn Ala Gly Ala 100 105
110 ggt acc aag gct gag taa 354 Gly Thr Lys Ala Glu 115 14 117 PRT
Aspergillus nidulans 14 Leu Pro Ala Ser Ala Ala Lys Asn Ala Lys Leu
Ala Thr Ser Ala Ala 1 5 10 15 Phe Ala Lys Gln Ala Glu Gly Thr Thr
Cys Asn Val Gly Ser Ile Ala 20 25 30 Cys Cys Asn Ser Pro Ala Glu
Thr Asn Asn Asp Ser Leu Leu Ser Gly 35 40 45 Leu Leu Gly Ala Gly
Leu Leu Asn Gly Leu Ser Gly Asn Thr Gly Ser 50 55 60 Ala Cys Ala
Lys Ala Ser Leu Ile Asp Gln Leu Gly Leu Leu Ala Leu 65 70 75 80 Val
Asp His Thr Glu Glu Gly Pro Val Cys Lys Asn Ile Val Ala Cys 85 90
95 Cys Pro Glu Gly Thr Thr Asn Cys Val Ala Val Asp Asn Ala Gly Ala
100 105 110 Gly Thr Lys Ala Glu 115 15 408 DNA Aspergillus nidulans
CDS (1)..(408) 15 atg cgc ttc atc gtc tct ctc ctc gcc ttc act gcc
gcg gcc acc gca 48 Met Arg Phe Ile Val Ser Leu Leu Ala Phe Thr Ala
Ala Ala Thr Ala 1 5 10 15 acc gcc ctc ccg gcc tct gcc gca aag aac
gcg aag ctg gcc acc tcg 96 Thr Ala Leu Pro Ala Ser Ala Ala Lys Asn
Ala Lys Leu Ala Thr Ser 20 25 30 gcg gcc ttc gcc aag cag gct gaa
ggc acc acc tgc aat gtc ggc tcg 144 Ala Ala Phe Ala Lys Gln Ala Glu
Gly Thr Thr Cys Asn Val Gly Ser 35 40 45 atc gct tgc tgc aac tcc
ccc gct gag acc aac aac gac agt ctg ttg 192 Ile Ala Cys Cys Asn Ser
Pro Ala Glu Thr Asn Asn Asp Ser Leu Leu 50 55 60 agc ggt ctg ctc
ggt gct ggc ctt ctc aac ggg ctc tcg ggc aac act 240 Ser Gly Leu Leu
Gly Ala Gly Leu Leu Asn Gly Leu Ser Gly Asn Thr 65 70 75 80 ggc agc
gcc tgc gcc aag gcg agc ttg att gac cag ctg ggt ctg ctc 288 Gly Ser
Ala Cys Ala Lys Ala Ser Leu Ile Asp Gln Leu Gly Leu Leu 85 90 95
gct ctc gtc gac cac act gag gaa ggc ccc gtc tgc aag aac atc gtc 336
Ala Leu Val Asp His Thr Glu Glu Gly Pro Val Cys Lys Asn Ile Val 100
105 110 gct tgc tgc cct gag gga acc acc aac tgt gtt gcc gtc gac aac
gct 384 Ala Cys Cys Pro Glu Gly Thr Thr Asn Cys Val Ala Val Asp Asn
Ala 115 120 125 ggc gcc ggt acc aag gct gag taa 408 Gly Ala Gly Thr
Lys Ala Glu 130 135 16 135 PRT Aspergillus nidulans 16 Met Arg Phe
Ile Val Ser Leu Leu Ala Phe Thr Ala Ala Ala Thr Ala 1 5 10 15 Thr
Ala Leu Pro Ala Ser Ala Ala Lys Asn Ala Lys Leu Ala Thr Ser 20 25
30 Ala Ala Phe Ala Lys Gln Ala Glu Gly Thr Thr Cys Asn Val Gly Ser
35 40 45 Ile Ala Cys Cys Asn Ser Pro Ala Glu Thr Asn Asn Asp Ser
Leu Leu 50 55 60 Ser Gly Leu Leu Gly Ala Gly Leu Leu Asn Gly Leu
Ser Gly Asn Thr 65 70 75 80 Gly Ser Ala Cys Ala Lys Ala Ser Leu Ile
Asp Gln Leu Gly Leu Leu 85 90 95 Ala Leu Val Asp His Thr Glu Glu
Gly Pro Val Cys Lys Asn Ile Val 100 105 110 Ala Cys Cys Pro Glu Gly
Thr Thr Asn Cys Val Ala Val Asp Asn Ala 115 120 125 Gly Ala Gly Thr
Lys Ala Glu 130 135 17 678 DNA Artificial Sequence Fusion protein
CDS (1)..(678) 17 atg aag atc acc gct gtc att gcc ctt tta ttc tca
ctt gct gct gcc 48 Met Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser
Leu Ala Ala Ala 1 5 10 15 tca cct att cca gtt gcc gat cct ggt gtg
gtt tca gtt agc aag tca 96 Ser Pro Ile Pro Val Ala Asp Pro Gly Val
Val Ser Val Ser Lys Ser 20 25 30 tat gct gat ttc ctt cgt gtt tac
caa agt tgg aac act ttt gct aat 144 Tyr Ala Asp Phe Leu Arg Val Tyr
Gln Ser Trp Asn Thr Phe Ala Asn 35 40 45 cct gat aga ccc aac ttg
aaa aag cgc ctc ccg gcc tct gcc gca aag 192 Pro Asp Arg Pro Asn Leu
Lys Lys Arg Leu Pro Ala Ser Ala Ala Lys 50 55 60 aac gcg aag ctg
gcc acc tcg gcg gcc ttc gcc aag cag gct gaa ggc 240 Asn Ala Lys Leu
Ala Thr Ser Ala Ala Phe Ala Lys Gln Ala Glu Gly 65 70 75 80 acc acc
tgc aat gtc ggc tcg atc gct tgc tgc aac tcc ccc gct gag 288 Thr Thr
Cys Asn Val Gly Ser Ile Ala Cys Cys Asn Ser Pro Ala Glu 85 90 95
acc aac aac gac agt ctg ttg agc ggt ctg ctc ggt gct ggc ctt ctc 336
Thr Asn Asn Asp Ser Leu Leu Ser Gly Leu Leu Gly Ala Gly Leu Leu 100
105 110 aac ggg ctc tcg ggc aac act ggc agc gcc tgc gcc aag gcg agc
ttg 384 Asn Gly Leu Ser Gly Asn Thr Gly Ser Ala Cys Ala Lys Ala Ser
Leu 115 120 125 att gac cag ctg ggt ctg ctc gct ctc gtc gac cac act
gag gaa ggc 432 Ile Asp Gln Leu Gly Leu Leu Ala Leu Val Asp His Thr
Glu Glu Gly 130 135 140 ccc gtc tgc aag aac atc gtc gct tgc tgc cct
gag gga acc acc aac 480 Pro Val Cys Lys Asn Ile Val Ala Cys Cys Pro
Glu Gly Thr Thr Asn 145 150 155 160 tgt gtt gcc gtc gac aac gct ggc
gcc ggt acc aag gct gag ctg gtt 528 Cys Val Ala Val Asp Asn Ala Gly
Ala Gly Thr Lys Ala Glu Leu Val 165 170 175 ccg cgt gga tcc atc gaa
ggt cgt ggc ggc cgc atc ttt tac cca tac 576 Pro Arg Gly Ser Ile Glu
Gly Arg Gly Gly Arg Ile Phe Tyr Pro Tyr 180 185 190 gat gtt cct gac
tat gcg ggc tat ccc tat gac gtc ccg gac tat gca 624 Asp Val Pro Asp
Tyr Ala Gly Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 195 200 205 gga tcc
tat cca tat gac gtt cca gat tac gct gct cag tgc ggc cgc 672 Gly Ser
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ala Gln Cys Gly Arg 210 215 220
taa tag 678 18 224 PRT Artificial Sequence Fusion protein 18 Met
Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10
15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser
20 25 30 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe
Ala Asn 35 40 45 Pro Asp Arg Pro Asn Leu Lys Lys Arg Leu Pro Ala
Ser Ala Ala Lys 50 55 60 Asn Ala Lys Leu Ala Thr Ser Ala Ala Phe
Ala Lys Gln Ala Glu Gly 65 70 75 80 Thr Thr Cys Asn Val Gly Ser Ile
Ala Cys Cys Asn Ser Pro Ala Glu 85 90 95 Thr Asn Asn Asp Ser Leu
Leu Ser Gly Leu Leu Gly Ala Gly Leu Leu 100 105 110 Asn Gly Leu Ser
Gly Asn Thr Gly Ser Ala Cys Ala Lys Ala Ser Leu 115 120 125 Ile Asp
Gln Leu Gly Leu Leu Ala Leu Val Asp His Thr Glu Glu Gly 130 135 140
Pro Val Cys Lys Asn Ile Val Ala Cys Cys Pro Glu Gly Thr Thr Asn 145
150 155 160 Cys Val Ala Val Asp Asn Ala Gly Ala Gly Thr Lys Ala Glu
Leu Val 165 170 175 Pro Arg Gly Ser Ile Glu Gly Arg Gly Gly Arg Ile
Phe Tyr Pro Tyr 180 185 190 Asp Val Pro Asp Tyr Ala Gly Tyr Pro Tyr
Asp Val Pro Asp Tyr Ala 195 200 205 Gly Ser Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala Ala Gln Cys Gly Arg 210 215 220 19 131 PRT Streptomyces
coelicolor 19 Met Leu Lys Lys Ala Met Val Ala Ala Ala Ala Ala Ala
Ser Val Ile 1 5 10 15 Gly Met Ser Ala Ala Ala Ala Pro Gln Ala Leu
Ala Ile Gly Asp Asp 20 25 30 Asn Gly Pro Ala Val Ala Asn Gly Asn
Gly Ala Glu Ser Ala Phe Gly
35 40 45 Asn Ser Ala Thr Lys Gly Asp Met Ser Pro Gln Leu Ser Leu
Val Glu 50 55 60 Gly Thr Leu Asn Lys Pro Cys Leu Gly Val Glu Asp
Val Asn Val Ala 65 70 75 80 Val Ile Asn Leu Val Pro Ile Gln Asp Ile
Asn Val Leu Ala Asp Asp 85 90 95 Leu Asn Gln Gln Cys Ala Asp Asn
Ser Thr Gln Ala Lys Arg Asp Gly 100 105 110 Ala Leu Ser His Val Leu
Glu Asp Leu Ser Val Leu Ser Ala Asn Gly 115 120 125 Glu Gly Arg 130
20 133 PRT Streptomyces coelicolor 20 Met Ile Lys Lys Val Val Ala
Tyr Ala Ala Ile Ala Ala Ser Val Met 1 5 10 15 Gly Ala Ser Ala Ala
Ala Ala Pro Gln Ala Met Ala Ile Gly Asp Asp 20 25 30 Ser Gly Pro
Val Ser Ala Asn Gly Asn Gly Ala Ser Gln Tyr Phe Gly 35 40 45 Asn
Ser Met Thr Thr Gly Asn Met Ser Pro Gln Met Ala Leu Ile Gln 50 55
60 Gly Ser Phe Asn Lys Pro Cys Ile Ala Val Ser Asp Ile Pro Val Ser
65 70 75 80 Val Ile Gly Leu Val Pro Ile Gln Asp Leu Asn Val Leu Gly
Asp Asp 85 90 95 Met Asn Gln Gln Cys Ala Glu Asn Ser Thr Gln Ala
Lys Arg Asp Gly 100 105 110 Ala Leu Ala His Leu Leu Glu Asp Val Ser
Ile Leu Ser Ser Asn Gly 115 120 125 Glu Gly Gly Lys Gly 130 21 112
PRT Agaricus bisporus 21 Met Ile Ser Arg Val Leu Val Ala Ala Leu
Val Ala Leu Pro Ala Leu 1 5 10 15 Val Thr Ala Thr Pro Ala Pro Gly
Lys Pro Lys Ala Ser Ser Gln Cys 20 25 30 Asp Val Gly Glu Ile His
Cys Cys Asp Thr Gln Gln Thr Pro Asp His 35 40 45 Thr Ser Ala Ala
Ala Ser Gly Leu Leu Gly Val Pro Ile Asn Leu Gly 50 55 60 Ala Phe
Leu Gly Phe Asp Cys Thr Pro Ile Ser Val Leu Gly Val Gly 65 70 75 80
Gly Asn Asn Cys Ala Ala Gln Pro Val Cys Cys Thr Gly Asn Gln Phe 85
90 95 Thr Ala Leu Ile Asn Ala Leu Asp Cys Ser Pro Val Asn Val Asn
Leu 100 105 110 22 119 PRT Agaricus bisporus 22 Met Val Ser Thr Phe
Ile Thr Val Ala Lys Thr Leu Leu Val Ala Leu 1 5 10 15 Leu Phe Val
Asn Ile Asn Ile Val Val Gly Thr Ala Thr Thr Gly Lys 20 25 30 His
Cys Ser Thr Gly Pro Ile Glu Cys Cys Lys Gln Val Met Asp Ser 35 40
45 Lys Ser Pro Gln Ala Thr Glu Leu Leu Thr Lys Asn Gly Leu Gly Leu
50 55 60 Gly Val Leu Ala Gly Val Lys Gly Leu Val Gly Ala Asn Cys
Ser Pro 65 70 75 80 Ile Thr Ala Ile Gly Ile Gly Ser Gly Ser Gln Cys
Ser Gly Gln Thr 85 90 95 Val Cys Cys Gln Asn Asn Asn Phe Asn Gly
Val Val Ala Ile Gly Cys 100 105 110 Thr Pro Ile Asn Ala Asn Val 115
23 32 DNA Artificial Sequence PCR primer 23 cagctgggtc tgctcgctct
cgtcgaccac ac 32 24 32 DNA Artificial Sequence PCR primer 24
gtgtggtcga cgagagcgag cagacccagc tg 32 25 30 DNA Artificial
Sequence PCR primer 25 gagggaacca ccaactgtgt tgccgtcgac 30 26 30
DNA Artificial Sequence PCR primer 26 gtcgacggca acacagttgg
tggttccctc 30 27 34 DNA Artificial Sequence PCR primer 27
taataactcg agatgcgctt catcgtctct ctcc 34 28 33 DNA Artificial
Sequence PCR primer 28 taataaggat ccttactcag ccttggtacc ggc 33 29
30 DNA Artificial Sequence PCR primer 29 ggtaccaagg ctgagctggt
tccgcgtgga 30 30 30 DNA Artificial Sequence PCR primer 30
tccacgcgga accagctcag ccttggtacc 30 31 36 DNA Artificial Sequence
PCR primer 31 attattccat ggctattagc ggccgcactg agcagc 36 32 30 DNA
Artificial Sequence PCR primer 32 gcctcaccta ttccactccc ggcctctgcc
30 33 30 DNA Artificial Sequence PCR primer 33 ggcagaggcc
gggagtggaa taggtgaggc 30 34 49 DNA Artificial Sequence PCR primer
34 taatttctcg agatgaagat caccgctgtc attgcccttt tattctcac 49 35 33
DNA Artificial Sequence PCR primer 35 gttgccgatc ctggtgtgct
cccggcctct gcc 33 36 33 DNA Artificial Sequence PCR primer 36
cacaccagga tcggcaactg gaataggtga ggc 33 37 30 DNA Artificial
Sequence PCR primer 37 aacttgaaaa agcgcctccc ggcctctgcc 30 38 35
DNA Artificial Sequence PCR primer 38 ggcagaggcc gggaggcgct
ttttcaagtt gggtc 35 39 552 DNA Aspergillus nidulans CDS (1)..(288)
CDS (508)..(549) intron (456)..(507) CDS (381)..(455) Intron
(289)..(380) 39 atg cgc ttc atc gtc tct ctc ctc gcc ttc act gcc gcg
gcc acc gca 48 Met Arg Phe Ile Val Ser Leu Leu Ala Phe Thr Ala Ala
Ala Thr Ala 1 5 10 15 acc gcc ctc ccg gcc tct gcc gca aag aac gcg
aag ctg gcc acc tcg 96 Thr Ala Leu Pro Ala Ser Ala Ala Lys Asn Ala
Lys Leu Ala Thr Ser 20 25 30 gcg gcc ttc gcc aag cag gct gaa ggc
acc acc tgc aat gtc ggc tcg 144 Ala Ala Phe Ala Lys Gln Ala Glu Gly
Thr Thr Cys Asn Val Gly Ser 35 40 45 atc gct tgc tgc aac tcc ccc
gct gag acc aac aac gac agt ctg ttg 192 Ile Ala Cys Cys Asn Ser Pro
Ala Glu Thr Asn Asn Asp Ser Leu Leu 50 55 60 agc ggt ctg ctc ggt
gct ggc ctt ctc aac ggg ctc tcg ggc aac act 240 Ser Gly Leu Leu Gly
Ala Gly Leu Leu Asn Gly Leu Ser Gly Asn Thr 65 70 75 80 ggc agc gcc
tgc gcc aag gcg agc ttg att gac cag ctg ggt ctg ctc 288 Gly Ser Ala
Cys Ala Lys Ala Ser Leu Ile Asp Gln Leu Gly Leu Leu 85 90 95
ggtacgtgat ccccactcag tcgctcccgg agaggctgag ggaagacgag cgacggtcta
348 gaaatggtgt gctaatagat gcatgtgtgc ag ctc tcg tcg acc aca ctg agg
401 Leu Ser Ser Thr Thr Leu Arg 100 aag gcc ccg tct gca aga aca tcg
tcg ctt gct gcc ctg agg gaa cca 449 Lys Ala Pro Ser Ala Arg Thr Ser
Ser Leu Ala Ala Leu Arg Glu Pro 105 110 115 cca acg tacgtctttc
agatctgcta caagtgaggc gatcaaaact aacatattcc ag 507 Pro Thr 120 tgt
gtt gcc gtc gac aac gct ggc gcc ggt acc aag gct gag taa 552 Cys Val
Ala Val Asp Asn Ala Gly Ala Gly Thr Lys Ala Glu 125 130 135 40 135
PRT Aspergillus nidulans 40 Met Arg Phe Ile Val Ser Leu Leu Ala Phe
Thr Ala Ala Ala Thr Ala 1 5 10 15 Thr Ala Leu Pro Ala Ser Ala Ala
Lys Asn Ala Lys Leu Ala Thr Ser 20 25 30 Ala Ala Phe Ala Lys Gln
Ala Glu Gly Thr Thr Cys Asn Val Gly Ser 35 40 45 Ile Ala Cys Cys
Asn Ser Pro Ala Glu Thr Asn Asn Asp Ser Leu Leu 50 55 60 Ser Gly
Leu Leu Gly Ala Gly Leu Leu Asn Gly Leu Ser Gly Asn Thr 65 70 75 80
Gly Ser Ala Cys Ala Lys Ala Ser Leu Ile Asp Gln Leu Gly Leu Leu 85
90 95 Leu Ser Ser Thr Thr Leu Arg Lys Ala Pro Ser Ala Arg Thr Ser
Ser 100 105 110 Leu Ala Ala Leu Arg Glu Pro Pro Thr Cys Val Ala Val
Asp Asn Ala 115 120 125 Gly Ala Gly Thr Lys Ala Glu 130 135 41 34
DNA Artificial Sequence PCR primer 41 taataaggat ccatgcgctt
catcgtctct ctcc 34 42 129 DNA Schizosaccharomyces pombe CDS
(1)..(126) 42 atg gac tca atg gct aac tcc gtt tct tcc tcc tct gtc
gtc aac gct 48 Met Asp Ser Met Ala Asn Ser Val Ser Ser Ser Ser Val
Val Asn Ala 1 5 10 15 ggc aac aag cct gct gaa act ctt aac aag acc
gtt aag aat tat acc 96 Gly Asn Lys Pro Ala Glu Thr Leu Asn Lys Thr
Val Lys Asn Tyr Thr 20 25 30 ccc aag gtt cct tac atg tgt gtc att
gca taa 129 Pro Lys Val Pro Tyr Met Cys Val Ile Ala 35 40 43 42 PRT
Schizosaccharomyces pombe 43 Met Asp Ser Met Ala Asn Ser Val Ser
Ser Ser Ser Val Val Asn Ala 1 5 10 15 Gly Asn Lys Pro Ala Glu Thr
Leu Asn Lys Thr Val Lys Asn Tyr Thr 20 25 30 Pro Lys Val Pro Tyr
Met Cys Val Ile Ala 35 40 44 27 DNA Schizosaccharomyces pombe 44
tataccccca aggttcctta catgtgt 27 45 135 DNA Schizosaccharomyces
pombe CDS (1)..(132) 45 atg gac tcc att gca act aac act cat tct tca
tcc att gtc aat gcc 48 Met Asp Ser Ile Ala Thr Asn Thr His Ser Ser
Ser Ile Val Asn Ala 1 5 10 15 tac aac aac aat cct acc gat gtt gta
aaa act caa aac att aaa aat 96 Tyr Asn Asn Asn Pro Thr Asp Val Val
Lys Thr Gln Asn Ile Lys Asn 20 25 30 tat act cca aag gtt cct tat
atg tgt gta att gct taa 135 Tyr Thr Pro Lys Val Pro Tyr Met Cys Val
Ile Ala 35 40 46 44 PRT Schizosaccharomyces pombe 46 Met Asp Ser
Ile Ala Thr Asn Thr His Ser Ser Ser Ile Val Asn Ala 1 5 10 15 Tyr
Asn Asn Asn Pro Thr Asp Val Val Lys Thr Gln Asn Ile Lys Asn 20 25
30 Tyr Thr Pro Lys Val Pro Tyr Met Cys Val Ile Ala 35 40 47 27 DNA
Schizosaccharomyces pombe 47 tatactccaa aggttcctta tatgtgt 27 48
126 DNA Schizosaccharomyces pombe CDS (1)..(123) 48 atg gac tca atg
gct aac act gtt tct tcc tcc gtc gtt aac act ggc 48 Met Asp Ser Met
Ala Asn Thr Val Ser Ser Ser Val Val Asn Thr Gly 1 5 10 15 aac aag
cct tct gaa act ctt aac aag act gtt aag aat tat acc ccc 96 Asn Lys
Pro Ser Glu Thr Leu Asn Lys Thr Val Lys Asn Tyr Thr Pro 20 25 30
aag gtt cct tac atg tgt gtc att gca taa 126 Lys Val Pro Tyr Met Cys
Val Ile Ala 35 40 49 41 PRT Schizosaccharomyces pombe 49 Met Asp
Ser Met Ala Asn Thr Val Ser Ser Ser Val Val Asn Thr Gly 1 5 10 15
Asn Lys Pro Ser Glu Thr Leu Asn Lys Thr Val Lys Asn Tyr Thr Pro 20
25 30 Lys Val Pro Tyr Met Cys Val Ile Ala 35 40 50 27 DNA
Schizosaccharomyces pombe 50 tataccccca aggttcctta catgtgt 27 51 9
PRT Schizosaccharomyces pombe 51 Tyr Thr Pro Lys Val Pro Tyr Met
Cys 1 5 52 586 DNA Aspergillus nidulans Intron (471)..(530) Intron
(338)..(389) 52 atgaagttct ccattgctgc cgctgtcgtt gctttcgccg
cctccgtcgc ggccctccct 60 cctgcccatg attcccagtt cgctggcaat
ggtgttggca acaagggcaa cagcaacgtc 120 aagttccctg tccccgaaaa
cgtgaccgtc aagcaggcct ccgacaagtg cggtgaccag 180 gcccagctct
cttgctgcaa caaggccacg tacgccggtg acaccacaac cgttgatgag 240
ggtcttctgt ctggtgccct cagcggcctc atcggcgccg ggtctggtgc cgaaggtctt
300 ggtctcttcg atcagtgctc caagcttgat gttgctggtc agttcttcga
aaatcacttt 360 cgtgatgccc caatgctaac aattaccagt cctcattggc
atccaagatc ttgtcaacca 420 gaagtgcaag caaaacattg cctgctgcca
gaactccccc tccagcgcgg tatgttccct 480 tgttttacag cttattcact
taaaccgatt aatctaacaa cgctcacagg atggcaacct 540 tattggtgtc
ggtctccctt gcgttgccct tggctccatc ctctaa 586 53 474 DNA Aspergillus
nidulans CDS (1)..(471) 53 atg aag ttc tcc att gct gcc gct gtc gtt
gct ttc gcc gcc tcc gtc 48 Met Lys Phe Ser Ile Ala Ala Ala Val Val
Ala Phe Ala Ala Ser Val 1 5 10 15 gcg gcc ctc cct cct gcc cat gat
tcc cag ttc gct ggc aat ggt gtt 96 Ala Ala Leu Pro Pro Ala His Asp
Ser Gln Phe Ala Gly Asn Gly Val 20 25 30 ggc aac aag ggc aac agc
aac gtc aag ttc cct gtc ccc gaa aac gtg 144 Gly Asn Lys Gly Asn Ser
Asn Val Lys Phe Pro Val Pro Glu Asn Val 35 40 45 acc gtc aag cag
gcc tcc gac aag tgc ggt gac cag gcc cag ctc tct 192 Thr Val Lys Gln
Ala Ser Asp Lys Cys Gly Asp Gln Ala Gln Leu Ser 50 55 60 tgc tgc
aac aag gcc acg tac gcc ggt gac acc aca acc gtt gat gag 240 Cys Cys
Asn Lys Ala Thr Tyr Ala Gly Asp Thr Thr Thr Val Asp Glu 65 70 75 80
ggt ctt ctg tct ggt gcc ctc agc ggc ctc atc ggc gcc ggg tct ggt 288
Gly Leu Leu Ser Gly Ala Leu Ser Gly Leu Ile Gly Ala Gly Ser Gly 85
90 95 gcc gaa ggt ctt ggt ctc ttc gat cag tgc tcc aag ctt gat gtt
gct 336 Ala Glu Gly Leu Gly Leu Phe Asp Gln Cys Ser Lys Leu Asp Val
Ala 100 105 110 gtc ctc att ggc atc caa gat ctt gtc aac cag aag tgc
aag caa aac 384 Val Leu Ile Gly Ile Gln Asp Leu Val Asn Gln Lys Cys
Lys Gln Asn 115 120 125 att gcc tgc tgc cag aac tcc ccc tcc agc gcg
gat ggc aac ctt att 432 Ile Ala Cys Cys Gln Asn Ser Pro Ser Ser Ala
Asp Gly Asn Leu Ile 130 135 140 ggt gtc ggt ctc cct tgc gtt gcc ctt
ggc tcc atc ctc taa 474 Gly Val Gly Leu Pro Cys Val Ala Leu Gly Ser
Ile Leu 145 150 155 54 157 PRT Aspergillus nidulans 54 Met Lys Phe
Ser Ile Ala Ala Ala Val Val Ala Phe Ala Ala Ser Val 1 5 10 15 Ala
Ala Leu Pro Pro Ala His Asp Ser Gln Phe Ala Gly Asn Gly Val 20 25
30 Gly Asn Lys Gly Asn Ser Asn Val Lys Phe Pro Val Pro Glu Asn Val
35 40 45 Thr Val Lys Gln Ala Ser Asp Lys Cys Gly Asp Gln Ala Gln
Leu Ser 50 55 60 Cys Cys Asn Lys Ala Thr Tyr Ala Gly Asp Thr Thr
Thr Val Asp Glu 65 70 75 80 Gly Leu Leu Ser Gly Ala Leu Ser Gly Leu
Ile Gly Ala Gly Ser Gly 85 90 95 Ala Glu Gly Leu Gly Leu Phe Asp
Gln Cys Ser Lys Leu Asp Val Ala 100 105 110 Val Leu Ile Gly Ile Gln
Asp Leu Val Asn Gln Lys Cys Lys Gln Asn 115 120 125 Ile Ala Cys Cys
Gln Asn Ser Pro Ser Ser Ala Asp Gly Asn Leu Ile 130 135 140 Gly Val
Gly Leu Pro Cys Val Ala Leu Gly Ser Ile Leu 145 150 155 55 420 DNA
Aspergillus nidulans CDS (1)..(417) 55 ctc cct cct gcc cat gat tcc
cag ttc gct ggc aat ggt gtt ggc aac 48 Leu Pro Pro Ala His Asp Ser
Gln Phe Ala Gly Asn Gly Val Gly Asn 1 5 10 15 aag ggc aac agc aac
gtc aag ttc cct gtc ccc gaa aac gtg acc gtc 96 Lys Gly Asn Ser Asn
Val Lys Phe Pro Val Pro Glu Asn Val Thr Val 20 25 30 aag cag gcc
tcc gac aag tgc ggt gac cag gcc cag ctc tct tgc tgc 144 Lys Gln Ala
Ser Asp Lys Cys Gly Asp Gln Ala Gln Leu Ser Cys Cys 35 40 45 aac
aag gcc acg tac gcc ggt gac acc aca acc gtt gat gag ggt ctt 192 Asn
Lys Ala Thr Tyr Ala Gly Asp Thr Thr Thr Val Asp Glu Gly Leu 50 55
60 ctg tct ggt gcc ctc agc ggc ctc atc ggc gcc ggg tct ggt gcc gaa
240 Leu Ser Gly Ala Leu Ser Gly Leu Ile Gly Ala Gly Ser Gly Ala Glu
65 70 75 80 ggt ctt ggt ctc ttc gat cag tgc tcc aag ctt gat gtt gct
gtc ctc 288 Gly Leu Gly Leu Phe Asp Gln Cys Ser Lys Leu Asp Val Ala
Val Leu 85 90 95 att ggc atc caa gat ctt gtc aac cag aag tgc aag
caa aac att gcc 336 Ile Gly Ile Gln Asp Leu Val Asn Gln Lys Cys Lys
Gln Asn Ile Ala 100 105 110 tgc tgc cag aac tcc ccc tcc agc gcg gat
ggc aac ctt att ggt gtc 384 Cys Cys Gln Asn Ser Pro Ser Ser Ala Asp
Gly Asn Leu Ile Gly Val 115 120 125 ggt ctc cct tgc gtt gcc ctt ggc
tcc atc ctc taa 420 Gly Leu Pro Cys Val Ala Leu Gly Ser Ile Leu 130
135 56 139 PRT Aspergillus nidulans 56 Leu Pro Pro Ala His Asp Ser
Gln Phe Ala Gly Asn Gly Val Gly Asn 1 5 10 15 Lys Gly Asn Ser Asn
Val Lys Phe Pro Val Pro Glu Asn Val Thr Val 20 25 30 Lys Gln Ala
Ser Asp Lys Cys Gly Asp Gln Ala Gln Leu Ser Cys Cys 35 40 45 Asn
Lys Ala Thr Tyr Ala Gly Asp Thr Thr Thr Val Asp Glu Gly Leu 50 55
60 Leu Ser Gly Ala Leu Ser Gly Leu Ile Gly Ala Gly Ser Gly Ala Glu
65 70 75 80 Gly Leu Gly Leu Phe Asp Gln Cys Ser Lys Leu Asp Val Ala
Val Leu
85 90 95 Ile Gly Ile Gln Asp Leu Val Asn Gln Lys Cys Lys Gln Asn
Ile Ala 100 105 110 Cys Cys Gln Asn Ser Pro Ser Ser Ala Asp Gly Asn
Leu Ile Gly Val 115 120 125 Gly Leu Pro Cys Val Ala Leu Gly Ser Ile
Leu 130 135
* * * * *
References